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dissertation entitled

GENETIC DETERMINANTS OF mRNA STABILITY
IN PLANTS

presented by

Mark Aikens Johnson

has been accepted towards fulfillment
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Ph.D. degree in Microbiology

 

 

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11/00 cJCIRcmatooue.pes-p.14

THE GENETIC DETERMINANTS OF mRNA STABILITY IN PLANTS
By

Mark Aikens Johnson

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Microbiology

Program in Cellular and Molecular Biology

1999

ABSTRACT
GENETIC DETERMINANTS OF mRNA STABILITY IN PLANTS
By

Mark Aikens Johnson

Regulation of mRNA stability is now known to be an important means of
controlling gene expression. Despite many exciting advances in recent years, there are
still many unresolved questions about the mechanisms responsible for mRNA
degradation. The aim of this thesis was to address how plant cells recognize unstable
messages and to understand the mechanisms the cell uses to degrade them. The work
presented here provides important information as well as a foundation for mechanistic
insights that will be made in the future.

In Section One, Sequence-Specific mRNA Degradation, the focus is on the DST
element, which targets certain plant transcripts for rapid mRNA degradation. Mutants of
Arabidopsis were isolated that do not fully recognize this instability signal. At least two
genes, dstl and d312, are shown to be involved in the recognition of the DST sequence.
The cloning of these genes will be of tremendous interest and will likely be very
informative about the recognition of unstable mRNAs. Also in this section, a dissection
of the SA UR-A CI 3’ untranslated region (UTR) is described. This 3’UTR contains one
DST element and several DST-like subdomains. Studies presented here indicate that
redundant DST-like sequences located upstream of the DST element may play an

important role in the instability fiinction of the SA UR-A C1 3’UTR. In the fiiture it will be

interesting to analyze the effect of these redundant DST-like sequences on mRNA
stability in the dstl and dst2 mutants.

In Section Two, General Mechanisms omeNA Degradation, two sets of
experiments address how the plant cell degrades mRNA. To get a glimpse into this
process, poly(G) tracts were inserted into reporter transcripts and these were introduced
into plant cells in an attempt to stabilize intermediates in the process of mRNA
degradation. Such intermediates were not observed, which may indicate that there are
some differences between the mechanisms of mRNA turnover in plants and yeast, where
poly(G) tracts are known to stabilize mRNA decay intermediates. The second set of
experiments addresses the role of a potential plant poly(A) ribonuclease, AtPARN, in
plant mRNA decay. It has been shown that removal of the poly(A) tail leads to rapid
mRNA degradation in yeast and mammals. Preliminary experiments indicate that
AtPARN has poly(A) ribonuclease activity in vitro, suggesting that it may play a role in

the degradation of mRNA in plants.

ACKNOWLEDGMENTS

I would like to thank Pam Green for being my mentor. The experiments
described in this thesis would not have been possible if not for her creativity, support,
guidance, encouragement, and enthusiasm. Pam has fostered a laboratory environment
that helps to make our work enjoyable and intellectually rewarding.

Many people have contributed to this work and while specific acknowledgements
are given at the end of each chapter there are a few people who deserve special thanks.
Miguel Pérez-Amador and I have worked together throughout the course of my Ph.D. He
has been an extremely helpful and caring colleague without whom the selection of dst
mutants would have been even more difficult. Likewise, Michael Sullivan was extremely
helpful when I first joined the laboratory and laid the groundwork for the mutant
selection. Linda Danhof has kept our laboratory well organized and has maintained an
army of undergraduate helpers. Jonathon Vogel, who started out in Linda’s army, has
made countless contributions to my work and has provided me with the pleasure of
helping him to learn how to do science. Preet Lidder and I worked together during her
rotation. She has and will continue to make important contributions to our study of the
dst mutants and to AtPARN. Thanks also to Ellen Baker and Jim Colbert who, along
with Pam, were my co-authors on the review article that became the introduction to this
thesis. I am also grateful for my thesis committee: Frans deBruijn, Donna Koslowsky,
and Mike Thomashow; who have taken time out of their schedules to give me excellent
advice at several important stages in this process.

Members of the Green Lab, past and present, have had a tremendous impact on

these experiments and on the way I think and communicate about science. They have

iv

been a great bunch of fiiends and colleagues. My thanks go to Jim Kastenmayer, Nikki
Lebrasseur, Michael Feldbriigge, Rodrigo Gutierrez, Gustavo MacIntosh, Mike Abler,
Pauline Bariola, Jay De Rocher, Scott Diehn, Pedro Gil, Christy Howard, Deb
Thompson, and Ambro van Hoof.

My thesis is dedicated to Carol, who has made this tremendous learning
experience a time of great joy for me, and to my entire family (my grand parents: Henry
and Virginia Aikens, Wilhelm and Hilda Johnson; my parents: Tom and Kathy Johnson,
Scott and Sherie Barnes; my brothers and sisters: Suzy, Jenny, Kate, and Andy Johnson,
and Bill Barnes; and all of my aunts, uncles, and cousins) who have been with me the

whole way.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................. x
INTRODUCTION ............................................................................................................... l
DETERMINANTS OF MESSENGER RNA STABILITY IN PLANTS
mRNA INSTABILITY SEQUENCES ........................................................................... 5
Instability Sequences in SA UR genes .......................................................................... 6
AUUUA sequences ................................................................................................... 10
Premature stop codons and other translational influences on mRNA stability ......... 11
Sequences involved in regulated mRNA degradation ............................................... 14
T RANS-ACTING FACTORS INVOLVED IN mRNA STABILITY ........................... 16
RNA binding proteins ............................................................................................... 16
RNA-degrading activities .......................................................................................... 18
The role of the cap and poly(A) tail in mRNA stability ............................................ 21
Poly(A)-binding proteins ........................................................................................... 24
PATHWAYS OF mRNA DEGRADATION ................................................................ 25
POST-TRANSCRIPTIONAL GENE SILENCING ..................................................... 31
FUTURE DIRECTIONS ............................................................................................... 33
THE SCOPE OF THIS THESIS ................................................................................... 34
REFERENCES .............................................................................................................. 37

SECTION I: SEQUENCE-SPECIFIC mRNA DEGRADATION

CHAPTER l-l .................................................................................................................. 49
A PROCEDURE TO SELECT MUTANTS OF ARABIDOPSIS DEFECTIVE IN
SEQUENCE-SPECIFIC mRNA DEGRADATION

INTRODUCTION ......................................................................................................... 50
RESULTS ...................................................................................................................... 53
Premise of the mutant selection strategy ................................................................... 53
Selection of transgenic plants to serve as the parental lines for mutagenesis ........... 58
Lines p1514-9 and p15 19-31 showed decreased hygromycin resistance ................. 62
Mutagenesis of lines p1514-9 and p15 19-31 ............................................................ 63
Selection of putative mutants .................................................................................... 64
DISCUSSION ............................................................................................................... 66
MATERIALS AND METHODS .................................................................................. 70
Plasmid construction and plant transformation ......................................................... 70
Plant material and growth conditions ........................................................................ 71
EMS mutagenesis and selection of hygromycin resistant plants .............................. 72
ACKNOWLEDGEMENTS .......................................................................................... 73
REFERENCES .............................................................................................................. 75

vi

 

CHAPTER 1-2 .................................................................................................................. 79
MUTANTS OF ARABIDOPSIS DEFECTIVE IN A SEQUENCE-SPECIFIC
mRNA DEGRADATION PATHWAY

REFERENCES AND NOTES ...................................................................................... 94
CHAPTER 1-3 .................................................................................................................. 98

ANALYSIS OF THE DST ELEMENT AND DST-LIKE SUBDOMAINS
WITHIN THE SA UR-A C1 3’ UNTRANSLATED REGION

INTRODUCTION ......................................................................................................... 99
RESULTS .................................................................................................................... 101
Mutagenesis of all DST and DST-like subdomains ................................................ 101
Gain of function analysis of DST and DST-like subdomains ................................. 105
DISCUSSION ............................................................................................................. l 10
MATERIALS AND METHODS ................................................................................ 115
Plasmid construction and plant transformation ....................................................... 115
Plant material and grth conditions ...................................................................... 117
RNA analysis ........................................................................................................... 117
AKNOWLEDGMENTS ............................................................................................. l 18
REFERENCES ............................................................................................................ l 19

SECTION 2: GENERAL MECHANISMS OF mRNA DEGRADATION

CHAPTER 2-1 ................................................................................................................ 121
USE OF POLY(G) IN SERTIONS TO GAIN INSIGHT INTO THE
STEPS INVOLVED IN PLANT rnRNA DEGRADATION

INTRODUCTION ....................................................................................................... 122
RESULTS .................................................................................................................... 125
DISCUSSION ............................................................................................................. 128
MATERIAL AND METHODS .................................................................................. 131
Plasmid construction and plant transformation ....................................................... 131
RNA analysis ........................................................................................................... 133
RNase H treatment .................................................................................................. 134
ACKNOWLEDGEMENTS ........................................................................................ 134
REFERENCES ............................................................................................................ 135

vii

CHAPTER 2-2 ................................................................................................................ 138
CLONING AND INITIAL CHARACTERIZATION OF A
POLY(A) RIBONUCLEASE FROM ARABIDOPSIS

INTRODUCTION ....................................................................................................... 139
RESULTS .................................................................................................................... 141
The RNaseD family and cloning of AtPARN ......................................................... 141
AtPARN is expressed throughout the plant ............................................................ 144
Characterization of AtPARN activity in vitro ......................................................... 145
DISCUSSION ............................................................................................................. 146
MATERIALS AND METHODS ................................................................................ 150
Cloning of AtPARN ................................................................................................ 150
Analysis of AtPARN expression .............................................................................. 151
Expression of AtPARN in E. coli ............................................................................. 151
TCA PARN assay .................................................................................................... 153
ACKNOWLEDGEMENTS ........................................................................................ 154
REFERENCES ............................................................................................................ 155

viii

LIST OF TABLES

INTRODUCTION

Table 1. Sequences that have been demonstrated to control

mRNA stability in plants. .................................................................................................... 6
SECTION I: SEQUENCE-SPECIFIC mRNA DEGRADATION

CHAPTER 1-1: A PROCEDURE TO SELECT MUTANTS OF ARABIDOPSIS
DEFECTIVE IN SEQUENCE-SPECIFIC mRNA DEGRADATION

Table l-l-l. EMS mutagenesis of transgenic Arabidopsis lines. .................................... 64

Table 1-1—2. Hygromycin selection experiments ............................................................. 65

ix

LIST OF FIGURES
INTRODUCTION
Figure 1. Critical regions of the DST sequence ................................................................. 8
SECTION 1: SEQUENCE—SPECIFIC mRNA DEGRADATION

CHAPTER l-l: A PROCEDURE TO SELECT MUTANTS OF ARABIDOPSIS
DEFECTIVE IN SEQUENCE-SPECIFIC mRNA DEGRADATION

Figure l-l-l. A strategy for selecting mutants defective in a sequence-specific mRNA

decay pathway. .................................................................................................................. 54
Figure 1-1-2. The basic structure of transgenes used to assess various reporters and

instability determinants in transgenic Arabidopsis calli. .................................................. 56
Figure 1-1-3. Accumulation of reporter transcripts in Arabidopsis ................................. 57
Figure 1-1-4. T-DNA constructs used in mutant selection strategy. ................................ 59
Figure 1-1-5. Southern blot analysis of candidate lines for mutagenesis ......................... 60
Figure 1-1-6. Northern blot analysis of candidate lines for mutagenesis ......................... 61
Figure 1-1-7. p1514-9 and p1519-3l Show increased hygromycin sensitivity ................. 62

CHAPTER 1-2: MUTANTS OF ARABIDOPSIS DEFECTIVE IN A SEQUENCE-
SPECIFIC mRNA DEGRADATION PATHWAY

Figure 1-2-1. Structure of transgenes and kinetics of degradation of reporter transcripts

used for dst mutant selection ............................................................................................. 84
Figure 1-2-2. The dst selection strategy. .......................................................................... 86
Figure 1-2-3. mRNA abundance of selected transcripts in dst] and dst2. ....................... 87
Figure 1-2-4. Analysis of HPH-DST mRNA stability in leaves

from p1519-31, dstI, and dst2 plants. ............................................................................... 91

CHAPTER 1-3: ANALYSIS OF THE DST ELEMENT AND DST-LIKE
SUBDOMAINS WITHIN THE SA UR-ACI 3’ UNTRANSLATED REGION

Figure 1-3-1. The SA UR-A C1 3’UTR contains one DST-element and DST-like

subdomains. ..................................................................................................................... l 02
Figure 1-3-2. The structure of chimeric genes used to test mutations

in the SA UR-A C1 3’UTR. ............................................................................................... 103
Figure 1-3-3. Mutations in the SA UR-A C1 3’ UTR affect polyadenylation of the Globin
reporter transcript. ........................................................................................................... 104
Figure 1-3-4. Structure of chimeric genes used to test individual subdomains within the
SA UR-A C1 3’UTR for instability function. .................................................................... 106
Figure 1-3-5. Analysis of the N, R, and D regions

of the SA UR-A C1 3’ UTR. .............................................................................................. 107
Figure 1-3-6. Analysis of combinations of N, R, and D regions .................................... 109

SECTION 2: GENERAL MECHANISMS OF mRNA DEGRADATION

CHAPTER 2-1: USE OF POLY(G) INSERTIONS TO GAIN INSIGHT INTO
THE STEPS INVOLVED IN PLANT mRNA DEGRADATION

Figure 2-1-1. The effect of a poly(G) insertion on the major yeast

mRNA degradation pathway. .......................................................................................... 125
Figure 2-1-2. Poly(G) tracts were inserted into a variety of
plant reporter transcripts .................................................................................................. 126

Figure 2-1-3. Poly(G) stabilized intermediates do not accumulate in Arabidopsis. ...... 128

CHAPTER 2-2: CLONING AND INITIAL CHARACTERIZATION OF A
POLY(A) RIBONUCLEASE FROM ARABIDOPSIS

Figure 2-2-1. Sequence alignment of the AtPARN and HuPARN

amino acid sequences ...................................................................................................... 143
Figure 2-2-2. AtPARN mRNA is found throughout the plant. ...................................... 144
Figure 2-2-3. AtPARN has poly(A) ribonuclease activity. ............................................ 146

xi

INTRODUCTION

DETERMINANTS OF MESSENGER RNA STABILITY IN PLANTS

In its original form, this introduction was published in “A Look Beyond Transcription:
Mechanisms Determining mRNA stability and Translation in Plants”, Julia Bailey-Serres,
Daniel R. Gallie, eds. Copyright 1998, American Society of Plant Physiologists (Johnson
et al., 1998). Changes have been made to update the content and bring the text into the

larger context of this dissertation.

INTRODUCTION

The abundance of an RNA molecule depends both upon the frequency with which
it is transcribed and the rate at which it is degraded. This statement holds true regardless
of whether one is considering mature mRNA molecules encoded by nuclear genes,
plastid-encoded mRNAs, nuclear-encoded rRNAS, or the abundance of a particular
species of primary transcript in the nuclear compartment. In each of these instances
degradative processes play an important role in the amount of RNA that is present, and
potentially, in the final level of gene expression.

The focus of this introduction will be on the degradative events affecting the
abundance of mature mRNAs encoded by nuclear genes in plants. It is generally
assumed that these types of degradative events occur within the cytosolic portion of the
cell. However, it also seems clear that nuclear degradative events may play a critical role
in the establishment of cytosolic mRNA levels in particular organs, tissues, or cells
(Kamalay and Goldberg, 1984; Okamuro and Goldberg, 1989). The significance of
mRNA degradation to gene expression is aptly demonstrated by attempts to express the
Bacillus thuringiensis toxin genes (cry genes) in transgenic plants. Wild-type versions of
cry genes are typically not expressed at high levels in transgenic plants (Adang et al.,
1993), even when fused to the strong cauliflower mosaic virus 358 (358) promoter. High
level expression has been obtained by use of synthetic, plant-like versions of the cry
genes that contain codon usage and other changes (Adang et al., 1993; Koziel et al.,
1993; Stewart et al., 1996; reviewed in Diehn et al., 1996). The plant-like cry genes are
generally thought to produce mRNAs that exhibit longer half-lives in plant cells (Diehn

et al., 1996), leading to higher level accumulation of the toxin. There is direct evidence

for this in the case of a plant-like synthetic cry] gene which encodes a transcript that is
considerably more stable than the wild-type crjyIA (c) transcript (De Rocher et al., 1998).

The half-lives of mRNA molecules can vary over a wide range. Typical plant
mRNA molecules appear to exhibit half-lives of several hours (Siflow and Key, 1979;
Sullivan and Green, 1993; Taylor and Green, 1995). Relatively unstable plant mRNAs,
with half-lives of an hour or less, have also been described (McClure and Guilfoyle,
1989; Braam and Davis 1990; Seeley et al., 1992; Taylor and Green, 1995; van Hoof and
Green, 1996). On the other end of the Spectrum are very stable mRNAs with half-lives of
days or more, some of which may be stored or sequestered from the mRNA decay
machinery. In addition, there are some well-characterized examples of mRNAs whose
half-lives can vary in response to specific stimuli. In mammalian cells, the transferrin
receptor mRNA is stabilized 20- to 30-fold under conditions of low intracellular iron
concentration (Casey et al., 1989; Koeller et al., 1991). Mammalian histone and B-
tubulin mRNAs also exhibit regulated variations in mRNA half-life (Ross, 1995). In
plants, a good example is the PvPRPI mRNA which is destabilized in the presence of
fungal elicitor (Zhang et al., 1993; Mehdy and Brodl, 1998).

There are a number of ways to measure mRNA decay rates in plant systems.
These include measuring the half-lives of endogenous or in vitro synthesized mRNAs in
protoplasts (Gallie et al., 1989) or in vitro (Byrne et al., 1993; Tanzer and Meagher,
1994). Alternatively, mRNA decay rates can be monitored in transformed or
nontransfonned cultured cells or intact plants, following treatment with a transcriptional
inhibitor (Newman et al., 1993; Byrne et al., 1993). Recently, repressible promoters have

also been used to shut off transcription of the genes of interest in cultured cells or plants

so that the stability of the corresponding mRNA can be measured without the use of
general transcriptional inhibitors (Weinman, et al., 1994; Gil and Green, 1996; Petracek
et al., 1998). For a more detailed description of these methods and a discussion of the
advantages and limitations of each, readers are referred to reviews by Abler and Green
(1996) and Ross (1995).

The refinement of methods for measuring mRNA stability, as well as mounting
evidence that many plant genes are regulated at this level, has led to considerable
progress in recent years. Information has been obtained about the contribution of the cap
and poly(A) tail, and it has been Shown that plants have multiple poly(A) binding
proteins that can be differentially regulated. Another important step has been the
identification of Specific sequences that control mRNA decay rates. In particular, several
sequences that cause rapid mRNA degradation have been characterized. These and other
studies have increased our perception of the types of cellular factors that may be involved
in mRNA decay and the mechanisms that may be involved. Finally, it has become
apparent that mRNA decay mechanisms may play a prominent role in certain forms of
gene silencing such as the phenomenon of post-transcriptional cosuppression that is
sometimes observed in transgenic plants. All of these topics will be discussed in this
introduction in an effort to present our current understanding of the components and
mechanisms that determine the inherent stabilities of different mRNAs in plants.
Emphasis will be placed on describing the most recent findings, many of which pertain to
rapid mRNA decay mechanisms that allow plants to respond quickly to internal and

external stimuli. The purpose of this introduction is to highlight what is known about

mRNA turnover in plants and to point out how the work presented in this dissertation

addresses some of the many important questions that are being addressed in this field.

mRNA INSTABILITY SEQUENCES

Rapid turnover of mRNA is thought to be an active process. Indeed, cis-acting
sequences have been identified that target transcripts for rapid turnover in plants as well
as in other systems (reviewed in Ross, 1995; Abler and Green, 1996; Caponigro and
Parker, 1996; van Hoof and Green, 1997). The DST element, found in the 3'
untranslated region (UTR) of the unstable small auxin up RNAS (SA URS), is one such
instability sequence that so far appears to be unique to plants (McClure et al., 1989;
Newman et al., 1993; Gil et al., 1994; Gil and Green, 1996; Sullivan and Green, 1996).
Another element that can target transcripts for rapid turnover in plants consists of repeats
of AUUUA pentamers (Ohme-Takagi et al., 1993). Multiple repeats of this pentamer are
a common feature of many highly unstable mammalian transcripts (reviewed in Chen and
Shyu, 1995). Premature stop codons have also been Shown to target transcripts for rapid
decay in plants as well as in yeast, Caenorhabditis elegans, and mammalian cells
(reviewed in Maquat, 1995; van Hoof and Green, 1997; Jacobson and Peltz, 1996). In
this section, the plant instability sequences that have been most thoroughly characterized

will be described. These are summarized in Table l.

 

Table 1. Sequences that have been demonstrated to control mRNA stability in plants.

 

 

 

 

 

 

Sequence Description Experimental System Ref
AUUUA AUUUA repeats are a common feature of t“; measured during 1
AU-rich instability determinants found in an ActD time course
labile mammalian cytokine and in stably transformed
protooncogene transcripts. l l repeats of BY-Z cells"
the AUUUA pentamer caused instability.

DST A highly conserved 40 base sequence tm measured during 2,3
found downstream of the stop codon in an ActD time course
unstable SAUR transcripts. A tandem in stably transformed
dimer of DST caused instability. BY-2 cells“

SA UR-ACI Approximately 140 bases, includes one tm measured using 4
3' UTR highly conserved DST element and ToplO promoter in
several repeated ATAGAT-like and stably transformed
GTA-like subdomains. BY-2 cells"
Premature Naturally occurring and in vitro t1 [2 measured during 5

Stop generated premature stop codons in the an ActD time course

Codon coding region of the PHA transcript. in stably transformed
BY-2 cells“

iLRE 5' UTR plus the first 14 codons of t”; measured 6
F ed-I ; controls light regulation of using ToplO
mRNA stability. promoter in

transgenic tobacco
plants

 

ActD, actinomycin D; UTR, untranslated region; Ref, References; PHA,
Phytohemagglutinin; BY-2, bright yellow-2 (NT-l, tobacco cell line); tug, half-life
References: l) Ohme-Takagi et al., 1993; 2) Newman et al., 1993; 3) Sullivan and
Green, 1996; 4) Gil and Green, 1996; 5) van Hoof and Green, 1996; 6) Petracek et al.,
1998) *Confirmed in transgenic plants by measrmg mRNA accumulation

Instability Sequences in SA UR genes

mRNAs encoded by the auxin induced SA UR genes are among the most unstable
plant transcripts and therefore offer an excellent model with which to study rapid mRNA
turnover. The half-lives of these transcripts have been estimated to be between 10 and 50
minutes depending upon the method used in the analysis (McClure and Guilfoyle, 1989;
Franco et al., 1990). Seven SA UR genes have been cloned from soybean, mungbean, and
Arabidopsis thaliana all of which contain a highly conserved, 40 base pair sequence

motif in their 3' UTRs (McClure et al., 1989; Yamamoto et al., 1992; Gil et al., 1994).

This sequence was termed DST because ofits location downstream of the SA UR stop
codon (McClure et al., 1989). The highly conserved nature of this sequence and its
placement in the 3' UTR of the SA UR transcripts led to the suggestion that the DST
element may control the stability of the SA UR transcripts (McClure et al., 1989; Franco et
aL,1990)

The destabilizing function of the DST element was tested directly by Newman et
a1. (1993). They demonstrated that a synthetic dimer of the DST element, when placed in
the 3' UTR of the B-glucuronidasc (GUS) or the ,BLglobin reporter transcript, is able to
target these reporter transcripts for rapid turnover relative to controls lacking DST
sequences. In these experiments, half-lives of reporter mRNAs were measured in stably
transformed tobacco (BY-2) cell suspension cultures by inhibiting new transcription
using actinomycin D and monitoring the decay of the reporter transcript over a two-hour
time course. It is likely that DST sequences act as potent instability determinants in
intact plants as well as in cultured cells because DST elements also cause a marked
decrease in mRNA abundance relative to controls in transgenic tobacco plants.

The DST element consists of three highly conserved subdomains separated by
two variable regions. Site-directed mutagenesis experiments have been performed in
order to determine which features of the DST element are most important for its fiinction
as an instability determinant (Sullivan and Green, 1996). The results of these
experiments are illustrated in Figure 1. Highly conserved subdomains are in boxes. The
second and third subdomains contain residues that are invariant among all DST elements
and are termed ATAGAT and GTA, respectively. Both of these subdomains have been

found to be necessary for DST function. This was shown by constructing mutated DST

elements in which either the ATAGAT or GTA subdomains were disrupted with five or
six base substitutions, respectively. These mutant elements were tested as dimers placed
in the 3' UTR of the ,B-globin reporter transcript. The effects of the mutations were
analyzed by measuring mRNA accumulation and half-life in stably transformed tobacco
cell cultures and mRNA accumulation in leaves of transgenic tobacco plants. These
mutations resulted in mRNA half-lives that were nearly as long as a control lacking DST
sequences and in the restoration of ,B-globin mRNA abundance to control levels in stably
transformed tobacco cell cultures and transgenic tobacco leaves. In order to pinpoint
critical bases within these two invariant subdomains, two base pair (CC) substitution
mutations were analyzed. The results indicated that the first four bases of the ATAGAT
subdomain are most important for DST function in tobacco cell culture. Interestingly, a
CC substitution in the invariant GTA sequence led to inactivation of the DST element in
transgenic tobacco leaves but not in cultured cells. This finding may indicate that the

DST element is recognized differently in different cell types.

Critical in all systems tested

 

 

 

[@6- - 5 - GETAGATTIG - 7 -|CAT_I'ITIIGTA I
‘-v-’ H“
Critical in both Critical in leaves
cultured cells but not
and leaves cultured cells

Figure 1. Critical regions of the DST sequence. DST sequences, found in unstable SA UR
transcripts, consist of three highly conserved regions (boxed) separated by two variable
regions. The second and third regions contain the invariant sequences ATAGAT and T--
GTA, respectively. Sequence requirements for function of the DST element were
established using a series of substitution mutations introduced into a dimer of the soybean
SA UR 15A DST element. All mutations were tested in both copies of the dimer.
Sequences shown to be critical using five and six-base substitution mutations are
highlighted above the sequence. Critical sequences identified with two-base pair
substitution mutations are highlighted below the sequence.

Although a function for SA UR gene products has not been demonstrated, the
expression patterns of these transcripts indicate that they may have a role in early events
in auxin-induced cell elongation. The SA UR-A C1 gene of A. thaliana has been carefiilly
analyzed in order to determine which regions of the gene are responsible for SA UR
expression characteristics, namely auxin induction and rapid mRNA turnover (Gil et al.,
1994; Gil and Green, 1996). By constructing chimeric genes that consisted of the SA UR-
ACI promoter, coding region, or 3' UTR fused to reporter genes, it was shown that the
promoter is the Site of auxin action, the 3' UTR is largely responsible for rapid mRNA
turnover and the coding region contributes to low mRNA abundance but not by
decreasing message half-life (Gil and Green, 1996). It was shown that the 3' UTR of
SA UR-ACI functions as a determinant of rapid mRNA turnover by fusing this region of
the SA UR gene to the B-globin coding region whose expression was driven by a
tetracycline-repressible promoter known as ToplO (Weinman et al., 1994). Following
tetracycline treatment, mRNA stability can be measured without the use of general
transcriptional inhibitors such as actinomycin D.

The SA UR-A C I 3' UTR contains one canonical DST element which is located 80
bases downstream of the stop codon and 10 bases upstream of the poly(A) addition site
(Gil et al., 1994). It is notable that experiments with the synthetic DST element,
described above, indicated that two copies were required for instability function. It is not
clear whether this apparent difference reflects an absolute DST recognition requirement
or if it reflects a necessary context within the SA UR-A C l 3' UTR. Interestingly, there are
several ATAGAT-like and GTA-like subdomains of the DST sequence located just

upstream of the classically defined DST element within the SA UR-A CI 3' UTR. These

redundancies may be serving as multiple recognition Sites for DST mediated mRNA

decay within the SA UR-A C1 3' UTR.

AUUUA sequences

AU-rich elements are found in the 3' UTRS of several of the most unstable
mammalian transcripts (Chen and Shyu, I995). The genes that encode these transcripts
have functions involved in cell division and differentiation and their expression is very
stringently controlled. Indeed, overexpression of some of these genes can lead to
oncogenesis and it is thought that AU-rich elements serve to limit expression by targeting
their transcripts for rapid decay. Repeats of the pentamer, AUUUA, are often found in
these AU-rich elements and have been shown to be important for their function (Caput et
al., 1986; Shaw and Kamen., 1986; Vakalopoulou et al., 1991; Shyu et al., 1991). A
repeat of eleven AUUUA pentamers has been shown to target reporter transcripts for
rapid degradation in plants (Ohme-Takagi et al., 1993). This was demonstrated by fusing
the repeated AUUUA element downstream of the flglobin or GUS coding region and
comparing the half-life of these transcripts to that of controls lacking AUUUA repeats
during actinomycin D time courses. The sequence of the element rather than its A+U
content appears to be most important because an AU-rich control lacking AUUUA
repeats had little effect on fl-globin or GUS mRNA stability.

These results indicate that the mRNA decay pathway mediated by AUUUA
repeats may be conserved between animals and plants. There is no evidence that this

pathway functions in yeast. The natural targets of the plant AUUUA mediated decay

10

pathway are unknown but one possible candidate is PvPRPI from Phaseolus vulgaris.
The PvPRPI transcript, which appears to encode a cell wall protein, is rapidly degraded
(t1/2s45') following the addition of fungal elicitor to bean cell cultures (Zhang et al.,
1993). Zhang and Mehdy have reported a 50 kd protein that can be specifically UV
cross-linked to the 3' UTR of the PvPRPI transcript (Zhang and Mehdy, 1994). The
binding of this 50 kd polypeptide, termed PRP-BP, has been mapped to a 27 bp sequence
that contains an AUUUA motif (Zhang and Mehdy, 1994). Although the 3' UTR of this
transcript has not been shown to be responsible for its rapid turnover, it is interesting to
consider the possibility that the PRP-BP is involved in the recognition of an AUUUA
motif that is involved in regulating the stability of this transcript. This speculation does,
however, beg the question of minimal sequence requirements for AUUUA repeats in
plants. Although this has not yet been established, recent data in mammalian cells
indicate that as few as three copies (Lagnado et al., 1994) or even one copy (Zubiaga et
al., 1994) of the sequence UUAUUUAUU are sufficient to destabilize a reporter

transcript.

Premature stop codons and other translational influences on mRNA stability

Premature stop codons function as cis-acting instability determinants in yeast, C.
elegans, mammalian cells and plants, and offer compelling evidence for a link between
translation and mRNA turnover that is widespread among eukaryotes (Maquat, 1995;
Jacobson and Peltz, 1996; Sullivan and Green, 1993; van Hoof and Green, 1997;

Marcotte, 1998). This was first recognized in plants when naturally occurring mutant

ll

alleles of phytohemagglutinin (PHA) and the Kunitz trypsin inhibitor were studied.
These alleles contain early stop codons and result in very little mRNA accumulation
(Voelker et al., 1986; Jofuku et al., 1989). Run-on transcription experiments showed that
the accumulation of the Kunitz trypsin inhibitor was being limited post-transcriptionally
(Jofuku et al., 1989). Recently, a study of the effects of premature stop codons in plants
has been conducted using the initially isolated PHA allele and other PHA alleles that
were constructed in vitro (van Hoof and Green, 1996). These experiments demonstrated
that premature stop codons, when present in the first 60% of the PHA coding region,
caused rapid mRNA turnover. This effect was measured by generating stably
transformed tobacco cell lines with mutant and control PHA alleles and monitoring
mRNA decay over an actinomycin D time course. mRNA accumulation studies carried
out in transgenic tobacco leaves confirmed that premature stop codons cause mRNA
instability in plants as well as in cell culture. Interestingly, a premature stop codon
placed 80% of the way through the coding region did not decrease mRNA stability in
stably transformed tobacco cells. One explanation for this finding is that mRNA turnover
is triggered by a certain length between start and stop codons or between the stop codon
and the poly(A) tail. This explanation is somewhat unsatisfying because coding regions
and 3' UTRS vary greatly in length and no correlation has been found between transcript
length and stability.

Alternatively, nonsense mediated mRNA decay in plants may require an
additional cis-acting element located downstream of the premature stop codon as has
been found in yeast. In yeast and in C. elegans, genetic analysis has led to the

elucidation of a pathway that is believed to have evolved to eliminate mRNAs with

premature stop codons so that the cell does not produce truncated and potentially harmful
polypeptides. This pathway is referred to as the nonsense mediated mRNA decay
pathway. In yeast, this pathway requires an early stop codon, a downstream cis-acting
element and trans-acting cellular factors (reviewed in Jacobson and Peltz, 1996). The
region between 60 and 80% of the PHA transcript may contain a cis-acting element that
is required for the function of the nonsense mediated decay pathway. This region does
not contain sequences similar to the downstream element that has been determined in
yeast (Zhang et al., 1995), however, it may contain a cis-acting element recognized by
plant nonsense mediated decay machinery.

The internal light regulatory element (iLRE) which comprises the 5' UTR and the
first 14 codons of the F ed-I transcript appears to be another cis-acting determinant of
mRNA stability that is closely linked to translation (Dickey et al., 1992). The pea Fed-I
gene encodes ferredoxin I which is a major photosynthetic electron carrier. Like other
nuclear encoded genes involved in photosynthesis, Fed-1 expression increases in
response to light. Levels of Fed-1 mRNA are about five fold higher in light-grown
versus dark-adapted plants (Elliot et al., 1989). The iLRE was identified because of its
ability to confer light responsiveness to reporter genes (Dickey et al., 1992; 1994). The
iLRE's location in the transcribed portion of Fed-1 and the observation that nuclear
run-on transcription experiments revealed no differences in transcription rates between
light-grown and dark adapted plants, provide a strong argument that the iLRE functions
post-transcriptionally at the level of mRNA stability (Dickey et al., 1992). Recently,
direct evidence that the iLRE modulates Fed-1 gene expression at the level of mRNA

stability was obtained using the ToplO promoter system in tobacco plants. This system

13

enabled Thompson and coworkers, to measure half-lives of reporter transcripts with or
without the iLRE in transgenic plants in light versus dark conditions (Petracek et al.,
1998). This system was also used to analyze the effects of 3-(3,4-dichlorophenyl)-1,1-
dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, on iLRE-
mediated mRNA degradation. The results of these experiments indicate that the
photosynthetic apparatus mediates light signaling that results in degradation of iLRE-
containing transcripts (Petracek et al., 1998). Previously it was shown that the iLRE
requires an open reading frame in order to modulate Fed-1 mRNA levels in response to
light, which indicates that control of F ed-I mRNA decay may be dependent upon
translation (Dickey et al., 1994). Further experiments have shown that iLRE-containing
messages are loaded on polyribosomes in illuminated plants but are on monosomes in the
dark (Dickey et al., 1998). Therefore it seems clear that translation plays a major role in
determining the stability of these transcripts. It would appear that active translation

protects the transcript from degradation in this case.

Sequences involved in regulated mRNA degradation.

PvPRPI and Fed-1, discussed above, are examples of genes that are regulated at
the level mRNA stability in response to a stimulus. There are many more reports in the
literature of message stability that responds to various stimuli including high fluences of
blue light (Anderson et al., 1999), cytokinin treatment (Downes and Crowell, 1998), and

heat shock (Belanger, et al., 1986). This mode of regulation provides the plant with a

14

means to rapidly tailor gene expression, which may be particularly important for plants
because they are firmly rooted in a dynamic environment.

Changes in (x-amylase message stability in response to sucrose starvation is a well
characterized example. The expression of several ()t-amylase genes is induced when rice
suspension cells are cultured without sucrose (Yu et al., 1991). Gene-specific probes
were designed that could differentiate between the eight members of this gene family and
it was shown by a combination of Northern blot analysis, nuclear run-on experiments,
and mRNA t( 1/2) analysis that three a-amylase genes are controlled at the level of
mRNA stability (Sheu et al., 1996). The 3’UTR of aAmy3 was studied in greater detail
and was shown to be largely responsible for the sugar regulation of this gene (Chan and
Yu, l998a, Chan and Yu, l998b). The aAmy3 3’UTR was divided into three sections
and each was introduced into the 3’UTR of the same reporter gene. Each construct was
introduced stably into rice cells and the effect on message stability was assayed during an
actinomycin D time course with or without the addition of sugar. Sections I and III were
shown to destabilize reporter transcripts in the presence of sugar (Chan and Yu, l998b).
This 3’UTR has a remarkable predicted secondary structure with several proposed stem-
loops. In fact, sections I and III are predicted to form stem-loops that have the same
sequence (AUAUAU) at the end of the loop (Chan and Yu, l998a). This may be just an
intriguing coincidence because the contribution of these sequences to message stability
has not been tested. However, there is a precedent for stem-loop structures being
important for regulated mRNA stability. The transferrin receptor of mammalian cells has
multiple stem-loops in its 3’UTR that serve as a binding site for the iron-responsive

element binding protein (IRE-BF). The IRE-BF is thought to protect the message from

15

degradation in iron limiting conditions. These same stem-loops appear to be the
recognition sequences for an endonuclease that cleaves the transcript when iron
concentration is high and the IRE-BP is no longer present (Mullner and Kuhn, 1988,

Casey et al., 1989, Binder et al., 1994).

T RANS-ACTING FACTORS INVOLVED IN mRNA STABILITY

Trans-acting factors involved in mRNA stability include both the actual
ribonucleases involved in the degradative process and regulatory factors which can be
supposed to act both positively (to stimulate rapid degradation) and negatively (to protect
from degradation). The close relationship between mRNA degradation and translation
for at least some mRNAs suggests that some factors may have dual roles in translation
and turnover (Jacobson and Peltz, 1996). There are three major approaches used in the
identification of new trans-acting factors: (1) cloning of genes encoding proteins with
known RNA-binding motifs and subsequent assignment of function; (2) identification of
proteins that bind to known cis-determinants of stability by gel mobility shift and/or
cross-linking assays; and (3) biochemical purification of proteins with ribonuclease

activity.

RNA binding proteins

The only factors with a likely role in stability to be identified by the first method

are the Arabidopsis poly(A)-binding proteins (Belostotsky and Meagher, 1993; 1996;

16

Hilson et al., 1993). Poly(A)-binding proteins (PABPs) from diverse organisms have a
characteristic set of four RNA-binding domains (RBDs) and a highly conserved region
which can be used as a tentative first identification (Burd and Dreyfuss, 1994). One of
the Arabidopsis PABPs (PAB5) has been further identified by its abilities to bind poly(A)
with high affinity (Belostotsky and Meagher, 1993) and to complement certain PABP
functions in yeast (Belostotsky and Meagher, 1996). The presumed function of the
PABPs in mRNA turnover is discussed below. A large number of other plant
RNA-binding protein cDNAs have been cloned and sequenced, using primers and probes
whose design was based on other related RBD-type sequences (e. g. see Guiltinan and
Niu, 1996 for a recent summary). To date, no definite functions have been assigned to
any of these proteins. Many are clearly not involved in cytoplasmic mRNA stability
because they localize to the chloroplast or nucleus. Since the great majority of yeast and
animal RED-containing proteins whose functions are known are involved in nuclear
processing and transport events (Burd and Dreyfuss, 1994), seeking RED-containing
proteins may not be the most effective way to uncover trans-acting factors involved in
turnover. At least ten RNP motifs have been distinguished (Mattaj, 1993), some of which
may turn out to be more prevalent in stability-related RNA-binding proteins.

The most direct approach to identifying trans-acting factors involved in stability
is to seek proteins that bind directly to, or participate in complexes which bind to, known
cis-acting stability determinants. For example, the iron response element binding protein
(IRE-BP) and a number of AU-rich element binding proteins in mammalian cells have
been identified in this way (e. g., Gillis and Malter, 1991; Rouault et al., 1989). Of

course, the identification of such proteins is a long step away from understanding how

17

they actually participate in degradation. Although a number of plant stability
determinants have been characterized (discussed above), no specific binding proteins
have been identified to date. The identification of a stability determinant does not
guarantee the future finding of a trans-acing factor, since not all stability determinants
necessarily function by serving as binding sites. One plant mRNA-binding protein which
may function in controlling stability is a 50-kD protein that binds to a sequence in the 3'
UTR of the bean PvPRPI mRNA (Zhang and Mehdy, 1994) The protein binding site, a
27-nt U-rich sequence, has not yet been demonstrated to be a determinant involved in this
destabilization event, but the protein binding activity in extracts increases afier elicitor
treatment, consistent with this scenario. Interestingly, the binding activity of the protein
is strongly influenced by its redox state. Redox regulation of RNA binding has been
described for a number of other proteins, some with known functions in the control of
stability and/or translation (Hentze et al., 1989; Malter and Hong, 1991; Chu et al., 1994),

and could turn out to be a widespread modus operandi for such regulators (Hentze, 1994).

RNA-degrading activities

Ribonucleases are expected to figure prominently among the protein factors that
participate in mRNA decay mechanisms. As discussed below, current models for mRNA
decay pathways in plants predict the involvement of both endo- and exoribonucleases,
although the relative importance of these two types of activities appears to differ
depending on the mRNA in question. Unfortunately, the plant RNases that we know the

most about are the least likely to be involved in mRNA degradation because these

18

enzymes enter the secretory pathway and are targeted to the extracellular space or
vacuole (see Bariola and Green, 1997, for a review). It should be noted that a role for the
vacuole in mRNA decay, particularly in the later stage of the process, can not be ruled
out because small fragments of RNA have been shown to exist in the vacuole (Abel et al.,
1990). However, it seems most likely that this RNA enters the vacuole by autophagy or
some other bulk process rather by mechanisms that target specific mRNAs to the vacuole
for degradation. The most common assumption is that mRNA decay mainly occurs in the
cytosol. The fact that translation is often coupled to mRNA decay supports this idea.
Whether some mRNAs are primarily degraded in the nucleus is, nevertheless, an open
question. Clearly RNA-degrading activities must exist in the nucleus to turnover introns
and 3' ends of precursor RNAS. Virtually nothing is known about these enzymes in any
system but it seems possible that some may be capable of degrading fully processed
transcripts prior to or during export.

Recently it was shown that RNase 111, an enzyme involved in pre-rRNA
processing as well as the decay of some specific mRNAs in Escherichia coli (Court,
1993), also participates in pre-rRNA processing in yeast nucleoli (Elela et al., 1996).
Examining plant genes with homology to RNase III (e. g. an Arabidopsis -expressed
sequence tag accession number 218464, Hofie et al., 1993) may be of particular interest
because most current models for antisense RNA-mediated inhibition (N ellen and
Lichtenstein, 1993) and some cosupression models argue for the involvement of a
dsRNase (discussed below). Other yeast activities known to be involved in mRNA decay
may also have plant homologues such the 5' to 3' exoribonuclease XRNI, which is a

major participant in the decay of most yeast mRNAs. Indeed, three XRN-like genes have

19

been cloned from Arabidopsis (James Kastenmayer and Pamela J. Green, unpublished).
XRNs have also been cloned from flies and mammals, indicating that these genes have
been conserved throughout eukaryotic evolution (Till et al., 1998, Bashkirov et al., 1997;
Shobuike et al., 1997). However. one potentially relevant observation is that insertion of
a poly(G) tract into an mRNA blocks the progression of XRNI in yeast giving rise to a
prominent intermediate that includes sequences 3' of the poly(G) tract (Vreken and Raué,
1992; Decker and Parker, 1993; Muhlrad et al., 1994). A 3' degradation intermediate also
results from poly(G) insertion into messages in Chlamydomonas, suggesting the
occurrence of a similar exonuclease activity in this organism (Gera and Baker, 1998;
Drager et al., 1998). Poly(G) intermediates have been looked for but not seen in both
higher plant and mammalian cell systems (Mark A. Johnson and Pamela J. Green,
Chapter 2-1 of this dissertation; Goddall, G., personal communication; Maquat, L.,
personal communication; Shyu, A.-B., personal communication) indicating that the role
of XRN—like genes may be less prominent, or different in higher eukaryotes. In any event,
additional efforts to characterize RNases that may participate in mRNA decay pathways
are certainly warranted.

As will be discussed in the next section, the 5’ cap and poly(A) tail play an
important role in determining mRNA stability. Therefore, it will be important to
characterize enzymes responsible for removing these structures during the process of
mRNA degradation. Recently, a poly(A) ribonuclease (PARN) was purified from calf
thymus which led to the isolation of a human cDNA encoding this enzyme (Komer and
Wahle, 1997; Komer et al., 1998). This is an intriguing activity because the enzyme

responsible for removing the poly(A) tail, the first step in the degradation of most yeast

20

mRNAs, has not yet been identified. An Arabidopsis PARN-like gene (AtPARN) has
been cloned and will be the subject of Chapter 2-2 of this dissertation. A decapping
complex has been described in yeast (Dunckley and Parker, 1999; Beelman et al., 1996).
There are also putative Arabidopsis homologues of these genes and the role that these
play in plant mRNA tum-over will be an interesting area of future work (Gustavo

MacIntosh and Pamela J. Green, unpublished).

The role of the cap and poly(A) tail in mRNA stability

A variety of primarily in vitro studies over many years have suggested a function
for the poly(A) tail and 7-methyl guanosine cap as mRNA-stabilizing structures
(reviewed in Baker, 1993; Stevens, 1993). However, the ability to definitively
demonstrate a stabilizing function in cells has been limited, in part, by an inability to
generate uncapped and unadenylated mRNA species in vivo. New genetic approaches in
yeast have finally conclusively demonstrated stabilizing functions for these sequences in
this organism. Decapping has been shown to be an early, sometimes rate-limiting, step in
the major degradation pathway of many mRNAs in yeast (Hsu and Stevens, 1993;
Muhlrad et al., 1994, 1995). Mutations that affect an essential component of the
decapping enzyme cause a marked stabilization of both normally unstable and stable
mRNAs (Beelman et al., 1996). In yeast, decapping is followed by rapid 5' to 3'
exonucleolytic digestion of the rest of the mRNA (by the XRNI nuclease discussed
below), and this sequence of events is likely to be the major degradative pathway for

yeast mRNAs. Because decapped mRNA is extremely labile in yeast, it could be

21

detected only in mutant cells with defective 5' to 3' exonuclease activity (xrnl cells) (Hsu
and Stevens, 1993; Muhlrad et al., 1994). The unavailability of similar mutants in other
organisms has so far precluded a demonstration of an analogous decay pathway. The
ability of an inserted tract of guanine nucleotides to trap 3' degradation intermediates by
impeding exonuclease progress has allowed the demonstration that the substrate for
decapping is, for many or most yeast mRNAs, a molecule which has undergone
substantial poly(A) shortening (Decker and Parker, 1993). Thus, a longer poly(A) tail
protects the mRNA from decapping, almost certainly via its ability to bind at least one
poly(A)-binding protein (Caponigro and Parker, 1995). A similar deadenylation-
dependent pathway has not yet been demonstrated to occur in other organisms. On the
other hand, observation of the poly(A) status of naturally stable degradation intermediates
has provided evidence against such a pathway for certain mRNAs (discussed below).
Alternative experimental options to directly test for stabilizing functions for the
cap and poly(A) tail in other organisms are limited. The best experimental approach to
date has been the study of in vitro transcribed mRNAs introduced into cells by
transfection, electroporation or injection. Only the evidence pertaining to plant cells will
be discussed here. Gallie and colleagues have conducted extensive analyses of the roles
of the cap and poly(A) tail in mRNA translation, using synthetic reporter mRNAs
electroporated into plant protoplasts (Browning et al., 1998). Comparisons of the
chemical and functional stability of capped versus uncapped and polyadenylated versus
unadenylated transcripts were included in some of these reports. Chemical stability was
measured simply by following the disappearance of the full length mRNA with time by

northern blot. A modest stabilizing effect of a 5' cap was observed: a cap increased the

22

half-lives of either unadenylated or polyadenylated luciferase transcripts about 2-fold,
from 31 and 44 minutes to 53 and 100 minutes, respectively. While all of these
electroporated transcripts can be considered relatively unstable, the rather small
stabilizing effect of a cap could be interpreted to mean that 5' to 3' exonuclease digestion
does not represent the major pathway for degradation of electroporated mRNAs.
Alternatively, it is possible that most capped transcripts are rapidly decapped following
delivery, and the small stability differential reflects the decapping rate. In either case, the
uncapped state does not dictate immediate degradation by a 5' to 3' exonuclease, since
uncapped mRNA is detectable for several hours. Given the extremely rapid degradation
that apparently follows decapping of natural mRNAs in yeast, it would be interesting to
know whether uncapped transcripts electroporated into yeast spheroplasts are subject to a
similar fate, or whether introduced transcripts are degraded by a different or slower
mechanism (Russell et al., 1991; Everett and Gallie, 1992). The addition of a 50-nt
poly(A) tail to electroporated reporter mRNAs was shown to also confer a modest 1.5 to
3-fold increase in chemical half-life (Gallie et al, 1989; Gallie, 1991). Again, it is not
clear how rapidly these reporter RNAS are deadenylated following delivery, or, in this
case, what fraction of delivered RNA acquires poly(A)-binding proteins (discussed
below). The stabilizing effects of a cap and poly(A) tail together are consistently
additive, unlike their contributions to translation, which are strongly synergistic.
Another possible explanation for the failure of a cap or poly(A) tail to act as
strong stabilizers of electroporated mRNAs is that the major substrates for putative
mRNase activities may be messenger RNPs, not naked RNA molecules. The extent to

which electroporated mRNAs associate with RNA-binding proteins is not known. It has

23

been determined, however, that only about 2% of electroporated mRNAs are polysomal
1.5 hours after delivery into carrot protoplasts (Gallie et al., 1995). It could be that only
this small subset of actively translated mRNAs are substrates for the usual mRNase
activities. This possibility was addressed by measuring the functional half-life of
electroporated mRNAs, defined as the time required to reach 50% of the final level of
protein produced. The functional half-life could be a more accurate measure of the
stability of those mRNAs that assemble into an mRNP form. In fact, when functional
half-lives were compared, the cap and poly(A) tail each did confer a somewhat stronger
stabilizing effect than their effects on chemical half-lives (3 to 5-fold versus 1.5 to
3-fold). However, the relatively modest protection suggests that the electroporated RNAS
are not substrates for potent deadenylation-dependent or decapping-dependent pathways
in these cells. It is not yet clear whether this finding reflects the absence of a dominant
role for the cap and poly(A) tail in plant mRNA stability or is the result of our inability to

evaluate the effect of these elements on the decay of transcripts synthesized in vivo.

Poly(A)-binding proteins.

Poly(A)-binding proteins (PABPs) are ubiquitous and multifunctional
RNA-binding proteins that have been implicated in several aspects of mRNA metabolism
and translation (reviewed in Jacobson and Peltz, 1996; Baker, 1997). The yeast
poly(A)-binding protein (pablp) has recently been assigned an unexpected stabilizing
function: it apparently prevents decapping of polyadenylated mRNAs, presumably in cis.

The evidence supporting this role is that decapping is normally triggered by shortening of

24

a poly(A) to a length too short to bind pablp (Decker and Parker, 1993; Muhlrad et al.,
1994) and in pabIA cells, decapping occurs without prior poly(A) shortening (Caponigro
and Parker, 1995). There are multiple PABPs in plants; in Arabidopsis, at least four
different PABP genes are expressed in a tissue and organ-specific manner (Belostotsky
and Meagher, 1993; 1996; Hilson et al, 1993). Whether these genes encode functionally
distinct or functionally redundant proteins is not yet known. To begin to address that
important question, Belostotsky and Meagher (1996) have expressed one of the
Arabidopsis PABP genes (PAB5) in yeast strains depleted of endogenous PABlp to
determine what functions could be complemented by the plant protein. Remarkably,
although the two proteins are only 44% identical at the amino acid level, the PABS
protein was able to rescue inviability and at least partially restore two functions: normal
poly(A) shortening and translation initiation. However, expression of the PARS gene did
not restore deadenylation-dependent decappin g of yeast mRNAs. Had it done so, this
would have strongly suggested the occurrence of a similar PABP function, and thus a
similar mRNA degradation pathway, in Arabidopsis. However, the inability of PABS to
complement this function in yeast does not imply that it lacks this function in
Arabidopsis. It will be important to see how other Arabidopsis PABPs behave in this

regard.

PATHWAYS OF mRNA DEGRADATION

Pathways by which mRNA molecules are degraded have been proposed for both

mammalian and yeast mRNAs. Mammalian mRNAs have generally been proposed to be

25

degraded in a 3' to 5' direction. Degradation of the body of message is thought to be
preceded by either stepwise shortening of the poly(A) tail (e.g., MYC mRNA), or by
endonucleolytic removal of the poly(A) tail (e.g., transferrin receptor mRNA) (Ross,
1996). Whether poly(A) removal is an essential rate-limiting step in the degradation of
mammalian mRNAs is not known, although the biphasic degradation kinetics of c-fos
and other ARE-containing mRNAs is compatible with that idea (Chen and Shyu, 1994).
In yeast, 3 major pathway of degradation appears to be the deadenylation-dependent
decapping pathway (Decker and Parker, 1993; Decker and Parker, 1994). After
decapping, degradation of some mRNAs (e. g., PGKl mRNA) appears to proceed
principally in the 5' to 3' direction. However, a more limited 3' to 5' degradation of the
FOX] mRNA has also been observed following deadenylation (Muhlrad and Parker,
1994). A deadenylation-independent decapping pathway also occurs (Muhlrad and
Parker, 1994). In this pathway, decapping and 5' to 3' degradation occur in the absence of ’
significant poly(A) shortening. mRNAs carrying premature nonsense codons, and
perhaps other mRNAs, are targeted for rapid degradation via this route. It would be
premature to conclude that yeast and mammalian cells degrade mRNAs by fundamentally
distinct pathways. An equally likely possibility is that eukaryotic cells have multiple
mRNA degradation pathways, with the pathway used being mRNA species specific. In
some species, one or more pathways may predominate and the predominant pathways

may differ among organisms.

26

Identification and analysis of in vivo produced mRNA degradation intermediates
would greatly facilitate the elucidation of mRNA degradation pathways. Unfortunately,
such intermediates are often not readily apparent in RNA samples from mammalian,
yeast, or plant cells. However, two plant mRNAs (encoded by the soybean SRS4 and oat
PH YA genes), for which putative in vivo mRNA degradation products have been
reported, have provided some insight into the pathways of mRNA degradation in plant
cells. It should be noted that mRNA degradation intermediates may be more prevalent
than is often assumed. It is common to observe hybridizing RNA fragments smaller than
the full-length mRNA in RNA blot analysis. Trimming of RNA blots prior to publication
to focus attention on the full-length band or to save journal space may obscure the true
frequency with which such RNA fragments occur. The problem, of course, is to
determine whether the RNA fragments are degradation intermediates produced in vivo, or
the result of degradation in vitro during isolation of the RNA samples. Control
experiments useful to address this question have been described (Seeley et al., 1992;
Thompson et al., 1992; Tanzer and Meagher, 1994).

A series of discrete fragments of the soybean ribulose-l ,5-bisphosphate
carboxylase small subunit (SRS4) mRNA are detected on RNA gel blots (Thompson et
al., 1992). Similar mRNA fragments are present in transgenic petunia transformed with
the soybean SRS4 gene under the control of the 35S promoter. These fragments appear to
be bona fide in vivo degradation intermediates. This interpretation is supported by the
observation that tracer RNAS are not degraded in homogenized samples during RNA
isolation (Thompson et al., 1992). In addition, degradation of in vitro synthesized SRS4

RNA in cell-free systems generates the same RNA fragments observed in vivo (Tanzer

27

and Meagher, 1994; 1995). The discrete nature of the SRS4 mRNA fragments suggests
the activity of an endonuclease. Sl nuclease and primer extension mapping of the SRS4
RNA fragments indicate that the fragments were indeed derived from endonucleolytic
cleavage of the full-length SRS4 mRNA (Tanzer and Meagher, 1995). The
endonucleolytic cleavage appears to be independent of both deadenylation and
decapping. Tanzer and Meagher (1995) have proposed that the endonucleolytic
cleavages are catalyzed by a stochastic endonuclease, cleaving at various sites within the
SRS4 mRNA, followed by 3' to 5' or 5' to 3' exonucleolytic cleavage of the resulting
fragments. The proximal (5') and distal (3') fragments would be degraded while
possessing either a 5' cap or poly(A) tail, respectively. As such, this model is distinct
from those proposed for mammalian and yeast mRNAs. Although, for example, a 5'
capped fragment of an mRNA could be thought of as being analogous to a deadenylated
mRNA, similar to deadenylation of mammalian transferrin receptor mRNA prior to
degradation (Ross, 1996).

RNA fragments have also been observed in RNA blot analysis of oat
phytochrome A (PHYA) mRNA. These fragments also appear to be in vivo degradation
products. This interpretation is based on several lines of evidence including the
observation that the PH YA fragments are present in RNA samples isolated by various
distinct procedures (Seeley et al., 1992). Other endogenous or exogenously-added tracer
RNAS remain intact under the same isolation conditions. Unlike the discrete fiagments
observed for soybean SRS4 mRNA, the PHYA fragments form a continuous distribution
ranging from full-length (approximately 4 kb) down to about 200 bp (Seeley et al, 1992;

Higgs and Colbert, 1994; Higgs et al, 1995). Analysis of these fragments, using distinct

28

probes corresponding to various regions of the PHYA mRNA molecule, has led to the
proposal that these fragments are generated by exonucleases (Hi ggs and Colbert, 1994).
RNase H mapping fails to reveal endonucleolytic cleavages near the 5' or 3' ends of the
mRNA (Higgs and Colbert, 1994). In addition, analysis of the polyadenylation status of
PHYA mRNA suggests that about 25% of the apparently full-length PH YA mRNA lacks a
poly(A) tail. PH YA RNA fragments are present in both the polyadenylated RNA
fraction and in the deadenylated RNA fraction. These observations have been
incorporated into a model (Hi ggs and Colbert, 1994) proposing that about 75% of the
PH YA mRNA population is degraded by a 5' to 3' exonuclease, with the poly(A) tail still
attached to the RNA fragment undergoing degradation. The deadenylated portion of the
PHYA mRNA population is proposed to be degraded by exonucleases acting at both the
3' and 5' ends of the molecule. If this model is correct, the bulk of the PHYA mRNA
would be degraded by a pathway similar to the deadenylation-independent decapping
pathway of yeast (Muhlrad and Parker, 1994).

The degradation pathway of mRNA molecules is a complex process about which
we have rather limited knowledge. It does seem clear that multiple pathways for mRNA
degradation may be present within eukaryotic cells. Even individual mRNA species may
be degraded by more than one pathway (Muhlrad and Parker, 1994; Higgs and Colbert,
1994). There are numerous unresolved questions regarding the pathway(s) of
degradation of nuclear-encoded mRNAs within plant cells. Perhaps the most pressing
basic question is the location of selective mRNA degradation. Two lines of evidence
suggest that degradation may occur on polysomes. First, putative in vivo degradation

products of both SRS4 mRNA and PHYA mRNA are present in polysomal RNA fractions

29

(Thompson et al., 1992; Byrne et al., 1993; Hi ggs and Colbert, 1994). Second, cell-free
mRNA degradation systems, based on isolated polysomes, have been described for both
plant (Byrne et al., 1993; Tanzer and Meagher, 1994) and animal (Brewer and Ross,
1990; Ross, 1995) cells. These cell-free systems appear to accurately reflect at least some
aspects of in vivo mRNA degradation. In addition, polysome-associated ribonuclease
activities have been reported (Green, 1994). However, other compartments within plant
cells are also known to possess ribonuclease activity. As discussed above, plant vacuoles
have high levels of ribonuclease activity (Abel and Glund, 1987; Green, 1994). The
possibility that some nuclear-encoded mRNAs are targeted for decay by regulated
transport into the vacuole is difficult to rule out. There is, however, only limited evidence
for the movement of RNA molecules across organellar membranes (Chang and Clayton,
1987; Oda et al., 1992).

Another area that warrants further development is the use of in vitro systems to
dissect mRNA decay pathways. Already, the two systems described above have been
shown to produce mRNA decay intermediates that mimic those observed in vivo. An
important next step will be to test whether cis-acting instability sequences can be
recognized in these and additional in vitro systems. Further characterization of the
process by which mRNA degradation occurs may also be possible using in vitro systems.
For example, recent evidence suggests that ATP is not required for the decay of PHYA
mRNA in vitro (Byrne and Colbert, unpublished). Clearly, additional exploration of
current in vitro systems and the development of new ones will provide further

mechanistic insights.

3O

POST-TRANSCRIPTIONAL GENE SILENCING

Cosuppression is a form of gene silencing that in many cases, appears to involve
degradation of the target mRNA. It was first identified during attempts by plant
molecular biologists to overexpress various cloned plant genes. These experiments
usually involved the generation of transgenic plants expressing the gene of interest driven
by the strong, constitutive, 358 promoter. Much to the surprise of these early
investigators, in a few of these transgenic lines, rather than over expression of the gene of
interest, both the transgene and the corresponding endogenous gene were coordinately
silenced (N apoli et al., 1990; van der Krol, 1990, Smith et al., 1990). This phenomenon
is not limited to transgenes with endogenous counterparts because it appears that multiple
copies of a transgene can suppress one another through similar mechanisms. In addition,
homology dependent virus resistance, a technique which employs portions of a viral
genome to generate transgenic plants that are resistant to the virus, may also function in
the same manner (Lindbo et al., 1993).

Many cases of cosuppression have been explained by transcriptional repression
that appears to be similar to other epi genetic phenomena described in fungi, plants, and
animals (Matzke and Matzke, 1996). In other cases, however, transcription rates of
transgenes and endogenous genes are normal, yet very little mRNA accumulates in the
cytoplasm. In these cases it has been assumed that expression is suppressed either by
blocking export of mature mRNA to the cytoplasm and eventual degradation in the
nucleus or by cytoplasmic mRNA degradation. Studies using cytoplasmically replicating

RNA viruses as targets for homology dependent virus resistance support the idea that

31

gene silencing is occurring in the cytoplasm via rapid degradation of the suppressed
mRNA (Lindbo et al., 1993). Further support for this idea comes from the finding that
nuclear mRNA abundance of a silenced transgene is unaffected (De Carvalho Niebel et
al,l995)

Post-transcriptional gene silencing (PTGS) of this type was once thought to be
limited to plants, but is now considered to be a mechanism that many organisms employ
to ward off invasive nucleic acid. The phenomena known as RNA interference and
quelling that have been studied in C aenorhabditis elegans and Neurospora crassa,
respectively, are probably examples of PTGS (Fire et al., 1998; Romano and Mancino,
1992). Most models that describe the initiation, maintenance, and spread of PTGS in
plants and other organisms call for the formation of a double-stranded RNA (dsRNA)
molecule. This is an attractive idea because it accounts for the sequence-specificity that
is observed in PTGS. This model implies the existence of an RNA-dependent RNA
polymerase (RDRP) that would be responsible for the synthesis of RNA complementary
to the target RNA and a dsRNA-specific RNase that would degrade the target RNA.
RDRPs have been cloned from plants and recently an RDRP was identified in a screen
for N. crassa mutants defective in quelling (Schiebel et al., 1998; Cogoni and Macino,
1999). RDRPs may generate small RNAS from specific target messages that would be
able to move from one cell to another within the organism to silence the target gene.
Recently evidence in support of this hypothesis has come by displaying small RNAS that
are complementary to silenced viral targets in plants (Hamilton and Baulcombe, 1999).
These studies represent dramatic progress in our understanding of how the PTGS signal

is generated. However, how dsRNA is degraded is still an open question. Insights into

32

these questions and other lingering questions about how PTGS works may come by
studying the mechanisms of rapid mRNA turnover. It is possible that the activities
involved in the final steps of both of these processes, namely rapid RNA degradation,

will be similar.

FUTURE DIRECTIONS

The development of reliable methods to measure mRNA half-lives in plants has
resulted in the identification of cis-acting sequences that target transcripts for rapid
turnover and in the identification of a trans-acting factor potentially involved in the
recognition of one of these elements. It is also now clear that alterations in gene
expression in response to extra and intra-cellular stimuli is in some cases controlled at the
level of mRNA stability in plants. In addition, understanding of the pathways involved in
mRNA turnover in plants has improved recently and points to possible differences
between plants and animals, and yeast. The future of this work must include increased
efforts aimed at the elucidation of mRNA degradation pathways and at identifying
cellular factors that carry out the steps in these pathways. By characterizing the decay of
a greater number of mRNAs, it will be possible to determine whether sets of mRNAs are
funneled into one or more distinct pathways and to determine whether there are any
common themes conserved among plants, fungi and animals. Molecular analysis of
cellular factors involved in mRNA degradation will improve efforts to characterize
mRNA turnover pathways and will provide insights into intriguing problems in plant

molecular biology such as post-transcriptional gene silencing.

33

The progress outlined in this introduction represents a foundation upon which a
rich future can be built. One exciting avenue of research will be the application of
molecular genetics in Arabidopsis thaliana in order to gain insight into the mechanisms
of mRNA turnover in plants and to identify cellular factors involved in this process.
Knowledge of cis-acting instability sequences should allow the isolation of mutants in
specific mRN A decay pathways. This may be particularly interesting in the case of AU-
rich elements because genetic approaches have been lacking in mammalian systems in
which the pioneering work on such elements has been done. Although most studies have
thus far dealt with unstable mRNAs, it is now possible to measure long mRNA half-lives
through the use of regulated promoters rather than prolonged incubation with
transcriptional inhibitors. This coupled with the finding of active stabilization
mechanisms in other systems opens the door for elucidating how the stabilities of long-
lived plant mRNAs are controlled. Genetic studies combined with biochemical
approaches aimed at purifying mRNA binding and degrading activities should lead to an
era of great expansion in our knowledge of fundamental mechanisms of mRNA

degradation in plants.

THE SCOPE OF THIS THESIS

The members of our laboratory we have begun to think of mRNA degradation as
a hierarchy consisting of three tiers (Gutierez et al., 1999). At the bottom of this scheme
are the basic mRNA degrading enzymes which dismantle and digest the message. Above

the basal mRNA degradation machinery is sequence-specific mRNA degradation which

34

determines the degradation rate of particular mRNAs. In this scheme, instability
determinants can be thought of as flags that target transcripts to the mRNA decay
machinery. Above sequence-specific decay is regulated mRNA degradation which is
more complex because the function of an instability sequence is determined by extra and
intracellular stimuli.

In my thesis, I have addressed the first two levels of this hierarchy and have
therefore divided this dissertation into two sections. In Section One, I report my work
aimed at understanding the mechanisms responsible for sequence-specific mRNA
degradation in plants. In Chapters 1-1 and 1-2 an approach is presented that resulted in
the isolation of two mutants that are defective in sequence-specific mRNA degradation
mediated by the DST element. In Chapter 1-3, I report on a series of experiments that
were designed to identify the critical instability sequences within the 3’UTR of SA UR-
AC1, an Arabidopsis transcript that is likely to be regulated by DST-mediated
degradation.

Section Two is devoted to my work on understanding the basic machinery
involved in degrading mRNA in plants. In a set of experiments described in Chapter 2-1,
a stable secondary structure was introduced into plant reporter transcripts in an attempt to
analyze intermediates in the mRNA decay process. In Chapter 2-2, preliminary work on
the characterization of a potential poly(A) ribonuclease of Arabidopsis is described.

The studies reported here represented steps along the way to answering some of
the important questions that have been laid out in this introduction. The major

contribution of this thesis is likely to be the dst mutants. These are the first mutants of

35

their kind to be isolated in any eukaryote and may be very useful in understanding the

mechanisms of sequence-specific mRNA degradation.

36

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48

CHAPTER 1-1

A PROCEDURE TO SELECT MUTANTS OF ARABIDOPSIS DEFECTIVE IN

SEQUENCE-SPECIFIC mRNA DEGRADATION

49

INTRODUCTION

Rates of messenger RNA (mRNA) degradation are highly variable in eukaryotic
cells (Reviewed in Ross, 1995; Caponigro and Parker, 1996; Johnson et al., 1998, also
see the introduction to this thesis). Some messages are degraded rapidly (minutes), while
others persist in the cell for much longer (days). These differences allow for precise
control of gene expression. It is important to know how the cell discriminates between
long and short-lived mRNAs if we are to fully understand how gene expression is
regulated.

Cis-regulatory elements have been identified that act as either instability or
stability determinants (for example, Holcik and Liebhaber, 1997; Chen and Shyu, 1995).
These elements have been defined by inserting them into messages of average stability
(half-life on the order of hours) and then measuring the half-life of the resulting chimeric
transcripts (Ross, 1995). Instability sequences target these otherwise stable reporters for
rapid degradation while stability elements stabilize the reporter mRNA. Our studies have
focused on messages that are very unstable because in many cases these messages encode
important regulatory proteins. This is logical because short-lived messages allow the cell
to achieve optimum control over gene expression in time and space, characteristics that
are important for the expression of key regulatory genes. Several sequences that target
transcripts for rapid turnover in plants have been identified. Among these are the DST
element (Newman et al., 1993), the AUUUA repeat (Ohme-Takagi, 1993), and premature

stop-codons (van Hoof and Green, 1996). We are interested in understanding the

50

mechanisms by which the plant cell recognizes these sequences and targets the transcripts
that contain them for rapid degradation.

Biochemical approaches have resulted in the isolation of sequence-specific RNA
binding proteins that are involved in modulating mRNA stability. The best example is
the iron-responsive element (IRE) which is bound by the IRE-binding protein (IRE-BP)
under iron-limiting conditions. Binding by the IRE-BP protects the transferrin mRNA
from degradation. The lRE-BP was purified from human liver by preparing biotinylated-
IRE RNA that was transcribed in vitro. A specific RNA binding protein was eluted from
biotin-agarose at high salt (Roualt et al., 1989). This led to the cloning of a cDNA for the
IRE-BP (Roualt et al., 1990). In addition, many RNA binding proteins have been
isolated that interact with AU-rich elements (ARE) in vitro. ARES are potent instability
determinants that result in rapid turnover of several mammalian proto-oncogenes and
often contain AUUUA repeats (Chen and Shyu, 1995). These experiments have relied on
UV-crosslinking and electrophoretic mobility shift assays to show that incubation of the
protein with a radiolabeled RNA probe results in a specific interaction. The challenge
has been to understand the contribution of these ARE-binding proteins to ARE-mediated
mRNA degradation in vivo.

Isolation of sequence-specific RNA-binding proteins from plant cells has not been
as successfiil perhaps because it is difficult to prepare cytoplasmic protein extracts that
are free of non-specific ribonucleases (F eldbrugge et al., unpublished data). This may be
due to the prominence of the plant vacuole, which is known to contain many non-specific
ribonucleases and other hydrolytic enzymes (Boller and Kende, 1979; Abel and Glund,

1987). Despite these difficulties, there is at least one example of a sequence-specific

51

RNA-binding—protein that may be involved in mRNA turnover in plants. The PRP-BP
binds specifically to a sequence in the 3’ untranslated region (UTR) of the Phaseolus
vulgaris proline rich protein (PvPRP) mRNA which is destabilized in response to fungal
ellicitor treatment of bean cell cultures (Zhang and Mehdy, 1994; Zhang et al., 1993).
The gene that encodes this protein and how it affects PvPRP mRNA stability are still
unknown.

An alternative to purification of sequence-specific RNA binding proteins would
be to isolate mutant plants that have lost the ability to recognize mRNA instability
determinants. This approach allows several important advantages. First, genes identified
in a mutant selection would be very likely to play a role in mRNA degradation in vivo.
Second, basic information about the mechanisms of mRNA degradation may be obtained
by studying mutants. Third, other cellular factors that might be involved in sequence-
specific interactions, such as RNA molecules, may be identified as long as they are
encoded by the nuclear genome. Finally, complications such as the presence of non-
specific RNA degrading activities in protein extracts are eliminated.

Threfore, parallel methods were developed to isolate mutants defective in DST-
and AUUUA-mediated mRNA degradation. There were several reasons to focus on
these instability determinants. The DST element has been well characterized and is likely
to play an important role in regulating the expression of many plant genes (Newman et
al., 1993; Sullivan and Green, 1996; Gil and Green, 1996). The AUUUA repeat contains
1 1 copies of the core sequence of the ARES, the most widely studied instability
determinant (Chen and Shyu, 1995, Lagnado et al., 1994; Zubinga et al., 1995). The

capacity to apply genetic analysis to ARE-mediated mRNA decay is limited in

52

mammalian cell cultures and this element is not known to function in model genetic
systems such as yeast. Therefore, studies in Arabidopsis may provide a unique
opportunity to study ARE-mediated mRNA degradation using mutants.

The selection strategy presented here is the first one designed to select mutants
that are defective in rapid mRNA degradation mediated by a specific sequence element.
This approach has resulted in the isolation of mutants defective in rapid mRNA
degradation mediated by the DST element (see Chapter 1-2). This procedure was based
on a selectable phenotype that we engineered using transgenic Arabidopsis plants and is
theoretically adaptable to any cis-regulatory element that results in rapid mRNA
degradation. Utilization of this method should lead to an increase in our understanding of
the basic mechanisms that are responsible for rapid degradation of mRNA in higher

plants.

RESULTS

Premise of the mutant selection strategy

In order to isolate mutants of Arabidopsis defective in a sequence-specific mRNA
degradation pathway we first needed to engineer plants that would display a mRNA
abundance phenotype that could be readily screened for and/or selected. This mutant
selection strategy relied on two reporter genes that each contained the DST or AUUUA
instability determinants in their 3’ UTR (Figure 1-1-1). In wild-type (wt) plants, the

instability determinant functions effectively and the abundance of the reporter mRNA is

53

maintained at a low level. Mutants that are defective in rapid mRNA degradation
mediated by the instability determinant would be expected to have an increased
abundance of both reporter mRNAs. If transgenes are chosen that encode readily
selectable and/or screenable enzymatic activities, it should be possible to isolate mutants

by virtue of an increase in reporter gene expression.

T-DNA

H mm

Parental Plants:
Low HPH mRNA
Hygromycin sensitive

 

Low GUS mRNA
Low GUS Activity

I; collect seed

4} mutagenlze

I} collect M2 seed
Mutants: _
High HPH mRNA 3; plate on hygromycin
Hygromycin resistant
High GUS mRNA
High GUS Activity

Figure 1-1-1. A strategy for selecting mutants defective in a sequence-specific mRNA
decay pathway. Transgenic plants are generated using a construct such as the one
represented above. Arrows represent promotor sequences. Hygromycin
phosphotransferase (HPH) and B-glucuronidase (GUS) are examples of reporter genes
that confer selectable and screenable phenotypes, respectively. I.D. represents the
instability determinant being studied, in this case either DST or AUUUA.

54

HPH and GUS are chosen as reporter genes

We initially considered hygromycin phosphotransferase (HPH), dihydrofolate
reductase (DHF R), and B-glucuronidase (GUS) as potential transgenes for this strategy.
HPH and DHF R confer resistance to hygromycin and methotrexate, respectively, and
there are many commercially available substrates that facilitate detection of GUS gene
expression (Nunberg et al., 1980; Gritz and Davies, 1983; Jefferson et al., 1987). The
impact of mRNA destabilization upon each of these selectable marker and reporter genes
was an important consideration. A large difference in mRNA abundance between plants
expressing a destabilized reporter versus a non-destabilized reporter would be optimal for
the isolation of mutants with a wider range of mRNA abundance phenotypes.

Previous experiments with transgenic tobacco plants showed that abundance of
the human ,B-Globin (Globin) transcript was reduced by ~14 fold when the AUUUA
repeat was inserted into its 3 ’UTR or by ~7.5 fold when a dimer of the DST element
(DSTx2) was inserted (Ohme—Takagi et al., 1993; Newman et al., 1993). In order to
assess the effect of AUUUA repeats and the DST element on the abundance of GUS,
HPH, and DHF R transcripts in Arabidopsis, T-DNA constructs were generated that
placed either the AUUUA repeat, DSTx2 or a tetramer of the DST element (DSTx4) into
the 3’UTR of each selectable marker or reporter gene. Similar constructs using the
Globin coding region were also analyzed so that the results in Arabidopsis could be
compared with those already obtained in tobacco. These constructs were designed to
have a reference gene within the same T-DNA to serve as a loading control for Northern

blot analysis. A representative construct is depicted in Figure 1-1-2.

55

Test

 

 

Reference

 

 

Figure 1-1-2. The basic structure of transgenes used to assess various reporters and
instability determinants in transgenic Arabidopsis calli. The cauliflower mosaic virus
35S promoter (35S) was used to control the expression of the DHFR, HPH, GUS, and
Globin reporter genes. Polyadenlytion signals derived from the pea ribulose 1,5-
bisphosphate carboxylase small subunit E9 (rch—E9, E9) gene were used as mRNA
processing signals for DHFR, HPH, and Globin. The 3’ UTR of the of the rch-3C (3C)
gene was used as a polyadenylation signal for GUS. The reporters were paired in test:
reference combinations as follows: DHFR with GUS, HPH with GUS (shown), GUS-
AUUUA with Globin, GUS-DST x2 with DHFR, GUS-DSTx4 with HPH, and Globin with
GUS. The right border (RB) of the transfer-DNA (T-DNA) has been indicated by a black
rectangle. The T-DNA also includes the neomycin phosphotranserase cassette so that
kanarnycin resistant transgenic tissue could be selected.

These constructs were used to generate transgenic Arabidopsis calli by co-
cultivating Agrobacterium strains harboring the relevant constructs with root explants of
Arabidopsis seedlings (Valveken eta1., 198 8). Pools of at least 100 independent
transgenic calli were gathered for each construct and the mRNA abundance of each
construct was analyzed by Northern blot analysis. Radiolabelled probes complementary
to the DHFR, HPH, GUS, and Globin coding regions were used to determine the
abundance of these reporter genes on blots that were loaded with equal amounts of total
RNA from pooled calli. Signals corresponding to reference and test transcripts were
quantitated by Phosphorlmager. The test signal for each construct was normalized using
the reference signal and the average normalized value was plotted relative to non-

destabilized control constructs (Figure 1-1-3).

56

DHFR HPH GUS Globin

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 ' " t r- P F I r-
75 I I n p
50 I I I p
25 I I I

:Control

- AUU UA repeat
rm DSTx2

:3 DSTx4

Figure 1-1-3. Accumulation of reporter transcripts in Arabidopsis. This histogram
depicts the normalized abundance of reporter transcripts in transgenic Arabidopsis calli.
Data are presented as a percentage of the non-destabilized controls; where multiple
experiments were done error bars have been plotted (standard deviation).

One important observation from this experiment was that neither a dimer of the
DST element (DSTx2) nor the AUUUA repeat destabilized the DHFR transcript to the
same extent as they did HPH, GUS, or Globin. It should be noted that the absolute
abundance of the DHFR transcript was lower than that of HPH, GUS, or Globin,
indicating that perhaps the DHF R transcript was relatively unstable to begin with (data
not shown). This might explain why insertion of instability sequences into this transcript
did not result in the same reduction in abundance that was observed for HPH, GUS, and
Globin. These results indicated that DHF R would not be an appropriate selectable
marker for our mutant selection strategy because an increase in mRNA abundance in a
putative mutant may not have been detectable above the already high relative abundance

of the transcript in wt plants. On the other hand, the observed effect of the AUUUA

repeat and the DST element on HPH and GUS mRNA abundance was consistent with

57

previously obtained Globin data and indicated that using these reporters would provide a

wider range in which to select new mRNA abundance phenotypes in mutants.

Selection of transgenic plants to serve as the parental lines for mutagenesis

HPH and GUS were chosen as the reporter genes for our selection strategy
(Figure 1-1-1). DSTx4 was used instead of DSTx2 because of the larger decrease in
mRNA abundance it caused. New constructs were made and then used to generate
transgenic Arabidopsis plants harboring both the selectable marker and reporter
transcripts destabilized by AUUUA (p1514) or DSTx4 (p1519). In addition, a positive
control construct was made that carried the same genes without instability determinants
(Figure 1-1-4). Since it was important to isolate plants with single inserts of the T-DNA
and that expressed the reporter genes to the appropriate level, several independent
transgenic lines were generated with each construct and these were examined to choose
the best lines for mutagenesis. Transgenic lines in which the reporter gene expression
reflected the average mRNA abundance found in the experiments shown in Figure 1-1-3,

became candidates to serve as the parental line for mutagenesis.

58

p1 14

HPH IAWUE
gas-Mus imam

 

 

 

 

 

 

 

 

Figure 1-1-4. T-DNA constructs used in mutant selection strategy. The T-DNAs also
include the neomycin phosphotranserase cassette so that kanamycin resistant transgenic
tissue could be selected. Also not shown is the nopaline synthase (nos) gene which contains
a BamHI restriction enzyme site and is located between the GUS-3 C gene and the right
border (RB).

In order to identify lines with one insert of the T-DNA, T2 (progeny of the self-
fertilization of the primary transforrnant) seed were plated on primary seed selection
medium containing 50 ug/ml kanamycin. Three p1519 (DSTx4) and three p1514
(AUUUA) lines that showed a ratio of three resistant to one sensitive seedling, indicating

inheritance of a single, dominant locus were chosen. Single inserts were confirmed by

Southern blot analysis (Figure 1-1-5).

59

p1514-9
p1514-22
p1514-21
- p1519-31
' p1519-26
p1519-13

-10Kb

‘ -fi'zu4Kb

In»
in! -- «In- an '1.2Kb

'AUUUA DSTx4

 

Figure 1-1-5. Southern blot analysis of candidate lines for mutagenesis. Genomic DNA
was extracted from transgenic plants, digested with BamHI, separated on a 1% agarose
gel, and transferred to a nylon membrane. The membrane was hybridized with a
radiolabeled probe complementary to the nopaline synthase (nos) coding region; adjacent
to the right border of the T-DNA. A 1.2 kb fragment that is part of the T-DNA is present
in all of the lanes. Additional bands represent the distance to BamHI sites in the genome.
The number of additional bands is indicative of the number of copies of the T-DNA.
p1514-9 and p1519-31 had simple banding patterns indicating that these lines had
single inserts. Northern blot analysis confirmed that each of these transgenic lines
expressed the HPH and GUS reporter transcripts to levels that were expected based on
previous experiments (Figure 1-1-6). Insertion of the AUUUA repeat into the 3’UTR of
the HPH or GUS transcripts resulted in an increase in mRNA size of 60 nucleotides.
Insertion of DSTx4 resulted in an increase of 180 nucleotides. Based on the results of this

experiment and those previously described, p1514-9 and pl 519-31 were chosen as the

parental lines for mutagenesis. The higher molecular weight GUS transcript (indicated

by (0) in Figure 1-1-6) that is present in the p1519-31 lane is a consistent characteristic of

60

this line. We chose p1519-31 for mutagenesis despite this because this line had the
simplest T-DNA insertion and because the additional band did not seem to alter GUS
activity (data not shown). We measured the abundance of the lower molecular weight

band, which is the expected size, throughout these experiments.

p1514—9

p1514-22
p1514-21
p1519-31
p1519-26
p1519-13

i<GUS

4 HPH

 

T AUUUA -DSTx4

 

Figure 1-1-6. Northern blot analysis of candidate lines for mutagenesis. 20 ug of total
RNA from l2-day-old seedlings was separated on a 1% agarose gel and transferred to a
nylon membrane. A. The membrane was simultaneously hybridized with radiolabeled
probes complimentary to the GUS and HPH coding sequences. B. A photograph of the
ethidiurn-bromide stained gel is shown indicating approximately equal loading of total
RNA.

61

Lines p1514-9 and p1519-31 showed decreased hygromycin resistance

In order for our selection strategy to be successfiil, the decrease in mRN A
abundance that resulting from the insertion of the AUUUA repeat or DSTx4 inserted into
the HPH transcript would have to result in decreased hygromycin resistance. In order to
test this, p1514-9, p1519-31, and p1493-2 seeds were plated on Arabidopsis germination
media containing 500, 750 and 100 ug/ml hygromycin. Seedlings were allowed to

germinate and grow for four weeks before examination (Figure 1-1-7).

p1493-2 p1514—9 p1519-31

g 1000
BI
a
.E
g 800
E
O
L
O?
>.
:: 500

 

+ AUU UA DSTx4

Figure 1-1-7. p1514-9 and p1519-31 show increased hygromycin sensitivity.
Approximately 500 seeds were surface sterilized and plated on primary seed selection
medium containing 50 ug/ml kanamycin plus the indicated concentration of hygromycin.
The photograph was taken after four weeks.

This experiment indicated that decreases in mRNA abundance due to insertion of

instability sequences into the HPH transcript caused increased hygromycin sensitivity in

62

p1514-9 and p1519-31 seedlings. This was especially clear at high concentrations of

hygromycin (1000 rig/ml) where very few seedlings escape this selection.

Mutagenesis oflines p1514-9 and p1519-31

Plants homozygous at the T-DNA locus present in lines p1514-9 and p1519-31
were obtained by plating T3 families on kanamycin and selecting those that segregated
100% resistance. Seed from multiple T3 plants were pooled so that adequate T4 seed
would be available for ethylmethane sulfonate (EMS) mutagenesis (Redei and Koncz,
1992, Li ghtner and Caspar, 1998). EMS is a standard mutagen for genetic analysis of
Arabidopsis. It is thought to cause point mutations primarily due to GC to AT transitions
that result from ethylation of DNA (Feldman et al., 1994). Standard EMS treatments
result in multiple, hemizygous mutations in each plant derived from mutagenized seed
(M.) such that only about 2000 M2 plants (self-fertilized progeny of M.) need be
analyzed to find a homozygous mutation in any one of the ~ 20,000 Arabidopsis genes
(Haughn and Somerville, 1986). We followed standard previously described EMS
mutagenesis protocols (Redei and Koncz, 1992). Table 1-1-1 describes the mutagenesis

ofp1514-9 and p1519-31.

63

 

Table 1-1-1. EMS mutagenesis of transgenic
Arabidopsis lines.
p1514-9 AUUUA
EXJ). # of MI Seed Group # of Ml/Group

 

 

 

Pilot 5000 Pilot 5000
1 100,000 1-20 5000
2 100,000 2 1-40 5000
3 50,000 41-52 4000
Total 255,000 4800

 

p1519-31 DSTx4
Exp. # of M1 Seed Group # of Ml/Group

 

 

 

1 100,000 1-20 5000
2 100,000 21-40 5000
3 25,000 41 -46 4000
Total 225,000 4900

 

Ml, first generation of mutagenized seed.

Following mutagenesis, M. seeds were planted in groups of ~5000 plants and M2
seed were collected from each group. M2 seeds were collected in groups in order to
obtain an initial approximation of the number of independent mutations, as mutants
selected from different groups were likely to represent independent mutations. The
effectiveness of EMS mutagenesis was evaluated by scoring the occurrence of albinism

and embryo lethality in MI plants, as previously described (Redei and Koncz, 1992).

Selection of putative mutants

Putative mutants were selected by plating ~4000 M2 seeds on primary seed
selection plates containing 1000 11ng hygromycin and 50 ug/ml kanamycin. After 3-4
weeks putative mutants, defined as seedlings that had developed at least two true leaves,

were chosen from the primary plates and moved to plates lacking hygromycin.

64

Development of two true leaves was consistently absent in non-mutagenized p1519-31
and p1514-9 seedlings. After one to two weeks on plates lacking hygromycin, putative
mutants were transferred to soil. Table 1-1-2 summarizes the results of the selection

experiments and the number of putative mutants that were obtained.

 

Table 1-1-2. Hygromycin selection
experiments
p1514-9 AUUUA
M2 group # M2 plated # putative

 

 

 

mutants
Pilot 4000 4
1-20 40,000 2
21-40 127,000 126
41-52 668,000 71
Total 839,000 203

 

p1519-31 DSTx4
M2 group # M2 plated # putative

 

 

 

 

mutants
1-20 90,000 14
2 l -40 444,000 263
41—52 200,000 46
Total 730,000 323
#, number; M2, second generation after

mutagenisis

The abundance of the HPH and GUS reporter mRNAs was determined by
Northern blot analysis of leaf RNA extracted from M2 plants selected on hygromycin.
These experiments allowed rapid scoring of the key mutant phenotype. The abundance
of the HPH and GUS transcripts was normalized using the translation initiation factor 4-
A (eIF4-A) transcript abundance; these were then compared to the normalized HPH and
GUS mRN A abundance of parental samples that had been grown in parallel. Where this
was not possible, for example if the plant was too small to obtain an adequate RNA

sample, mRNA abundance was assayed in M3 plants. In addition, M3 seeds were plated

65

on hygromycin to determine whether the resistance phenotype was inherited.
Hygromycin resistance was found to be heritable in approximately 20% of the M3
families tested, suggesting a high rate of false positives.

323 putative mutants derived from mutagenesis of line p1519-31 (DSTx4) and
203 derived from p1514-9 (AUUUA) were analyzed by these criteria. mRNA abundance
was analyzed by Northern blot analysis for all putative mutants. Only three putative
mutants met the criteria of heritable increases in HPH and GUS mRNA abundance.
These were DST-M2#64 (group 21), DST-M2#l 14 (group 31), and DST-M2#290 (group
30). No mutants were isolated from the AUUUA population even though over 800,000
p1514-9 M2 seeds were plated. DST-M2#114 and DST-M2#64 were isolated relatively
early in the selection process and were characterized in detail. These mutations were
found to be in independent genes and have been renamed dstI (DST-M2#1 14) and dst2
(DST-M2#64) to reflect their defects in DST-mediated mRNA degradation. A detailed
characterization of these mutants is the subject of the next chapter of this thesis. DST-
M2#290 is currently being analyzed. The immediate priorities are to establish the mode
of inheritance of the mutation and to determine whether this mutant complements either

dstI or dst2.

DISCUSSION

Genetic selection strategies such as the one presented here have been called
second generation screens or ‘targeted genetics’ because they rely on engineered

phenotypes rather than those intrinsic to the plant (for example, see Hooley, 1998).

66

Targeted genetics can provide a novel class of mutants to an area where classical screens
have already been saturated. The recently isolated age (guxin-responsive gene
expression) mutants provide an excellent example (Oono et al., 1998). Using a fusion of
an auxin-responsive promoter element to the GUS reporter gene, Oono et al., were able to
isolate auxin response mutants that misexpressed the reporter. These mutants provide a
useful complement to the many auxin-response mutants that have been identified by
traditional means.

Perhaps more importantly, targeted genetics can provide an entry into areas that
have been inaccessible to genetic analyses because obvious, intrinsic phenotypes are
lacking. This was the case for sequence-specific mRNA degradation mediated by
AUUUA repeats or the DST element. No targets of the AUUUA-mediated mRNA
degradation pathway have been definitively identified in plants. Therefore, it is difficult
to imagine an intrinsic phenotype that would result from the disruption of this pathway.
The DST element is likely to be involved in the constitutive instability of several
Arabidopsis SA UR transcripts. Therefore, it may be possible to devise a direct screen for
mutants based on the abundance of these transcripts. However, this would be extremely
labor-intensive and many of the mutants would be defective in the transcriptional
regulation of these genes by auxin. In fact, auxin-response mutants have been isolated
that result in misexpression from the SA UR-A C1 promoter (Leyser et a1. 1996). It is for
these reasons that a targeted genetic approach was necessary in order to explore the
genetic basis for sequence-specific mRNA degradation in plants.

The mutants isolated in this study were very rare. Three were isolated from a

total of approximately 1.5 x106 mutagenized seeds that were plated during the course of

67

the DST and AUUUA mutant selections. One of the possible explanations for this
observation is that genes involved in AUUUA- and DST-mediated mRNA degradation
are essential. Since it is likely that mRNA degradation pathways limit the expression of
key regulatory genes, it is possible that if transcripts targeted by these pathways are
overexpressed or misexpressed during early development of the plant due to a mutation,
the result would be embryo lethality. A relevant and interesting observation is that
overexpression of AUUUA-containing oncogene messages due to mutations that delete
AREs have resulted in neoplastic transformation in mammalian cells (Piechaczyk et al.,
1985). This finding highlights the potential consequences that can result when mRNA
degradation pathways are rendered ineffective by mutations.

If these pathways are essential for development of plants, one might also expect
redundancy to have evolved in these systems. This is another reason why mutants in the
AUUUA pathway may not have been isolated. As is described in the next chapter of this
thesis, dstI and dst2 were partially dominant mutations. If the DST-mediated mRNA
degradation pathway is redundant, then one might expect to only uncover dominant
alleles that interact with and negate redundant members of the pathway. Mutations in the
ethylene receptor illustrate how dominant negative alleles can provide access to
redundant genes (reviewed by Theologis, 1998). The original ethylene receptor
mutations were all dominant. After a receptor gene was isolated by map based cloning, it
was found that ethylene receptors constitute a gene family in Arabidopsis. Loss-of-
function mutant phenotypes were not observed until triple and quadruple mutants were

constructed (Hua and Meyerowitcz, 1998).

68

There are also technical explanations for the observed rarity of sequence-specific
mRNA degradation mutants. Approximately 80% of the putative mutants that were
chosen for further study were found to be false positives. This may have been due to
overzealous picking of putative mutants. However, this may have been important
because the mutants that were recovered, dstl and dst2, showed only slight increases in
HPH and GUS mRNA abundance and may have been missed if only the healthiest plants
were rescued from selection plates. In any case, this rate of false-positives, decreased the
efficiency of this selection.

Targeted genetics approaches have been used previously to isolate mutations that
affect the function of specific promoter sequences (Susek et al., 1993; Bowling et al.,
1994; Jackson etal., 1995; Martin et al., 1997; Oono et al., 1998). Here, the utility of
such a procedure has been extended to cis-regulatory elements located in the 3’UTR.
Therefore, the mutants isolated using this procedure have the potential to provide insight
into rapid sequence-specific mRNA degradation in plants and eukaryotes in general and
the cloning of dst genes may aid in the ellucidation of the molecular machinery that
catalyzes the degradation of DST-containing transcripts. These studies have the potential
to answer many fundamental questions about mRNA turnover in plants. For example, do
DST-recognition components recruit the basal mRNA decay machinery more rapidly to
DST-containing transcripts? One may find that dst gene products interact with poly(A)
binding-proteins and/or poly(A) ribonucleases so that the poly(A) tails of DST-containing
messages are removed more rapidly. The basic mechanisms of mRNA degradation in

plants are still largely unknown. Any insight into factors that interact with dst gene

69

products may reveal clues that will lead to the basic components responsible for this

process.

MATERIALS AND METHODS

Plasmid construction and plant transformation

Standard molecular cloning procedures were used (Sambrook et al., 1989).
Construction of the chimeric genes described in Figure 1-1-2 and Figure 1-1-4 was as
described by Newman et al., 1993. Chimeric genes were constructed in pBluescript
SKII+ (Stratagene) or pUC (New England Biolabs) and were then transferred to
derivatives of pMON505 which is a binary vector that facilitates transformation of plants
via Agrobacterium tumefaciens (Agrobacterium)(Rogers et al., 1987, Fang et al., 1989).
Chimeric genes had the basic structure: SacI-35S-BglII-XbaI-Coding region-BamHI-
poly(A) sequence-Clal. DST and AUUUA sequences were inserted at the BamHI site
between the coding sequence and the poly(A) signal. The source of the Globin and GUS
coding regions has been described (Newman et al., 1993). The HPH coding region was
obtained as a BamHI fragment of pLG90 (Gritz and Davies, 1983). The DHFR coding
sequence was described by Nunberg et al., (1980). The DST sequence used in these
experiments is the same as that found in the soybean SA UR-I5A gene and was
synthesized as described by Newman et a1, 1993. The construction of the AUUUA repeat

was described by Ohme-Takagi et al., 1993. The use of the 35S promoter and

70

polyadenylation signals from the 3’ ends of the E9 and 3C genes was as described by
Newman et al., 1993.

Transgenic Arabidopsis calli were generated by co-cultivation of Agrobacterium
with root explants (Valveken et al., 1988). A grobacterium strain LBA4404 was
transformed with pMON505 derivatives by electroporation using the Gene-Pulser
(BioRad) according to the manufacturer’s guidelines. Generation and selection of
transgenic Arabidopsis plants using the vacuum infiltration method of Agrobacterium-

mediated transformation has been described (N. Bechtold et al.,1993 and Web site:
http://www.bch.msu.edu/pamgreen/vac.htm). Agrobacterium strain GV3101 C58C1 Rif
(pMP90) (Koncz and Schell, 1986) was transformed with pMON505 derivatives by

electroporation using the Gene-Pulser (BioRad) according to the manufacturer’s

guidelines.

Plant material and growth conditions

The Columbia (Col-O) accession of Arabidopsis thaliana was used in all
transformations except for p1514 and p1519. To avoid any hygromycin resistant
contamination ofp1514-9 and p1519-31 during large-scale selection experiments, these
constructs were introduced into a Columbia mutant that lacks trichomes (glI, Col-PRL).
Arabidopsis calli were maintained on Gamborg’s BS (An, 1985) plates in an incubator set
at 16 hours light (125 pE/mz) /8 hours dark and 21°C. Arabidopsis plants were grown in
standard Arabidopsis soil in controlled environment growth chambers set at 16 hours

light (125 uE/mz) /8 hours dark, 21°C.

71

EMS mutagenesis and selection of hygromycin resistant plants

Seeds homozygous at the p1514-9 and p1519-31 loci (fourth generation of self-
fertilized progeny following transformation) were soaked for 16 hours in a 0.3% (v/v,
water) solution of EMS. Following mutagenesis, seeds were washed extensively with
water over a 12-hour period and then sown on soil. A thorough protocol is given in
Li ghtner and Caspar, (1998). The effectiveness of EMS mutagenesis was evaluated by
scoring the occurrence of albinism and embryo lethality in M1 plants as described by
Redei and C. Koncz (1992); and Li ghtner and Caspar, (1998).

Seed selection media consisted of 4.3 g/L Murashige and Skoog salt mixture
(GibcoBRL), BS vitamin mixture (100 mg/l myo-inositol, 10 mg/l thiamine
hydrochloride, 1 mg/l nicotinic acid, 1 mg/l pyriodoxine), 1% sucrose, 0.5 g/L MES at
pH 5.7, 0.8% phytagar, 1000ug/ml hygromycin and 50 ug/ml kanamycin. Hygromycin

was omitted when selecting transgenic plants or scoring inheritance of the T-DNA.

RNA analysis

RNA was isolated from transgenic calli (Figure 1-1-3) and pooled seedlings
(Figure 1-1-6) using the method described by Puissant and Houdebine (1990) with
modifications described by Newman et al., (1993). For analysis of mRNA abundance in
individual plants, two rosette leaves were harvested from mature plants and RNA was
extracted using the method described by Verwoerd et a1. (1989). Northern blot analysis

was as described by Newman et a1. (1993). Hybridization probes corresponding to the

72

DHFR, eIF4-A, Globin, GUS, and HPH coding regions were labeled using 32P dCTP by
the random-primed method of F einberg and Vogelstein ( 1983). The translation initiation
factor, eIF4-A, was used as normalization standard for northern blots as described by
Taylor et al., (1993). mRNA abundance was quantitated by measuring the intensity of

radioactive bands on Northern blots using a Molecular Dynamics Phosphorlmager.

DNA analysis

Southern blotting was performed as described in Sambrook et al., 1989. DNA
was extracted from dark-grown seedlings using the method described by Saghai-Maroof
et a1. (1984). ISug of DNA was digested with BamHI and loaded on a 1% agarose gel
which was transferred to a nylon membrane. Prehybridization, hybridization, and
washing conditions were the same as those for Northern blots (Newman et al., 1993).
Hybridization probes corresponding to the nos coding region were labeled using 32P

dCTP by the random-primed method of Feinberg and Vogelstein (1983).

ACKNOWLEDGEMENTS

I am grateful to Dr. Michael L. Sullivan who constructed the chimeric genes used
in this study and got me started on this project. Dr. Sullivan and Debrah M. Thompson
generated the data presented in Figure 1-1-3. The selection of putative mutants in the
AUUUA pathway was mainly the work of Miguel A. Pérez-Amador. I would also like to

thank Linda Danhof and Jonathan Vogel for tireless technical assistance. This work was

73

fiinded by grants from the United States Department of Energy, the United States

Department of Agriculture, and the McKnight Foundation to Pamela J. Green.

74

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78

CHAPTER 1-2

MUTANTS OF ARABIDOPSIS DEFECTIVE IN A SEQUENCE-SPECIFIC

mRNA DEGRADATION PATHWAY

This manuscript has been submitted for publication (Mark A. Johnson, Miguel A. Pérez-

Amador, and Pamela J. Green).

79

ABSTRACT

One of the ways a cell can rapidly and tightly regulate gene expression is to target
specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate
rapid mRNA decay have been identified in various eukaryotes, but the nature of the
cellular components that play critical roles in sequence-specific decay in vivo has been
more difficult to establish. Here, we used a novel genetic strategy to isolate rare mutants
of Arabidopsis that selectively elevate the abundance of mRNAs that contain the plant
instability sequence DST. Analysis of these mutants identified two loci that are
important for DST-mediated mRNA decay and provided a powerful inroad into the
mRNA decay machinery. Similar strategies should be applicable to the in vivo analysis

of sequence-specific mRNA decay in other eukaryotes.

80

The small-auxin-up-RNA (SA UR) transcripts are among the most unstable
mRNAs in plants, having half-lives of 10—50 minutes (1). The SA UR genes have been
implicated in auxin-induced cell elongation and their transcripts appear to be
constitutively unstable so that their abundance can be rapidly altered in response to
transcriptional control by the plant hormone auxin. All unstable SA UR transcripts
contain conserved DST sequences, so called because of their location (_lowns_tream of the
coding region (1). A dimer of the DST element (2) or the 3’ end of the Arabidopsis
SA UR-A C1 gene, which contains a DST element (3), are sufficient to destabilize reporter
transcripts in plant cells. The DST sequence is approximately 45 nucleotides in length
and is comprised of three highly conserved domains separated by two variable regions.
Mutagenesis studies have demonstrated that residues within two of the conserved
domains are necessary for instability function (4). Our detailed understanding of the DST
element, its robust effect on mRNA levels, and its potential biological importance make it
an attractive model for understanding how mRNA instability sequences function in
plants. Although genetic strategies have proven critical for the identification of
components of the general decay machinery in yeast (5), they have rarely been applied to
the study of rapid mRNA decay mediated by specific sequences. Most in vivo studies of
the latter have been limited to overexpression of RNA binding proteins (6) or correlations
between target mRNA levels and the presence of various RNA binding proteins (7).
However, it was recently demonstrated that mice made deficient for tristetraprolin by
gene disruption were defective in rapid degradation of tumor necrosis factor-0t mRNA
mediated by an AU-rich element (ARE). It was later shown that tristetraprolin binds

ARES in vitro (8). ARES are perhaps the most well studied class of mRNA instability

81

sequence in multi-cellular eukaryotes (9). Therefore, these results highlight how a single
mutant can provide important insights about sequence-specific mRNA decay mechanisms
in eukaryotes.

We have devised an approach to isolate mutants of Arabidopsis thaliana that are
defective in the sequence-specific mRNA decay pathway mediated by the DST element.
Because this strategy facilitated the isolation of rare mutations, it provided us with a
unique opportunity to study a sequence-specific mRNA degradation pathway in vivo.
Our approach was based on a phenotype that we engineered using two DST-containing
reporter genes that were introduced into Arabidopsis via Agrobacterium tumefaciens
mediated transformation (10). These reporter genes were designed to allow selection of
mutants that elevate expression of these genes due to defects in DST-mediated mRNA
degradation. Plasmid 1519 (p1519), used to generate the parental line for mutagenesis,
contained a tetramer of the DST element inserted into the 3’ UTR of the hygromycin
phosphotransferase (HPH) and B—glucuronidase (GUS) genes (Figure 1-2-l-A). HPH
confers resistance to hygromycin and GUS expression is readily detected by incubation of
plant tissue with substrates that are cleaved to colorimetric or fluorescent products (1 1).
We used a tetramer of the DST element because previous experiments had indicated that a
greater decrease in reporter mRNA abundance was achieved relative to a dimer (12). This
was beneficial because it provided a greater range of potential elevated mRNA abundance
mutant phenotypes. p1493 lacks the DST sequence and p1493-2 plants served as a non-
destabilized control throughout our experiments (Figure 1-2-1-A). In addition to the HPH
and GUS reporter genes, the T-DNA contained the nptII (neomycin phosphotransferase)

cassette so that kanamycin resistant transgenic plants could be selected. Transgenic line

82

p1519-31 was chosen for mutagenesis because segregation of kanamycin resistance and
Southern blot analysis indicated that it harbored a Single insert of the T-DNA (13). In
addition, the abundance of the HPH-DST and G US-DST messages in this line reflected
the average abundance of these transcripts in a population of p1519 transforrnants (13).
The location of the T-DNA insertion in p1519-31 was mapped to chromosome 11 near
position 51 (14).

The DST tetramer resulted in an approximately 10 fold decrease in steady-state
HPH mRNA abundance in plants harboring p1519 compared to those transformed with
p1493 (1 3). The half-life of the HPH transcript was reduced by approximately 3 fold in
p1519-31 plants relative to p1493-2 as measured in an actinomycinD time-course conducted
on 12-day-old seedlings grown in liquid culture (Figure l-2-l-B and l-C). The disparity
between the effect of the DST sequence on the steady-state abundance and the half-life of
the HPH transcript (10 fold vs. 3 fold) is most likely due to dampening that is commonly

observed when general inhibitors of transcription are used to measure mRN A decay rates

(15).

83

A
p1519 p1493

exam-Ia R. €91 13cm

 

0' 30’ 60’ 90’ 120150:
p1519-31(4)

 

hph mRNA 0
Remaining

 

 

 

.‘i‘fifi 91493‘21') 0.1 ; ; . ;

T

O 30 60 90 120150

Time (min)

Figure 1-2-1. Structure of transgenes and kinetics of degradation of reporter transcripts
used for dst mutant selection. (A) Diagrams representing the plant transformation
vectors, p1519 and p1493 are Shown. p1519 was used to generate transgenic Arabidopsis
plants that served as the parental line for the dst mutant selection. The cauliflower
mosaic virus 358 promoter was used to control the expression of the HPH and GUS
genes (28). Polyadenylation Signals were derived from the 3’ ends of the pea ribulose
1,5-bisphosphate carboxylase small subunit E9 (rch-E9, E9) and rch-3C (3C) genes.
The HPH and GUS transcripts encoded by p1519 have been destabilized by a tetramer of
the DST sequence (DSTx4). p1493 was used to generate transgenic plants that served as
a non-destabilized control during the mutant selection and in subsequent experiments.

(B) Comparison of the stability of HPH transcripts in parental seedlings (p1519-31) and
non-destabilized, control seedlings (p1493-2). Seedlings were grown in liquid culture for
12 days. ActinomycinD (75 ug/ml) was added, samples were removed from the cultures,
and frozen in liquid nitrogen at 30 minute intervals. Northern blot analysis was
performed with 10 ug of total RNA extracted from each sample (29). The blot was
hybridized with a radiolabeled probe complementary to the HPH transcript. The blot
displaying the p1519-31 time course has been overexposed relative to the p1493-2 blot so
that the Signal at all time points would be visible. (C) Signals obtained from Northern
blots were quantitated by Phosphorlmager (Molecular Dynamics) and plotted on a semi-
log scale. Linear regression analysis is Shown for each set of points.

84

Destabilization of the HPH transcript by the DST element resulted in reduced
resistance to hygromycin in p1519-31 seedlings relative to p1493-2 seedlings, particularly at
higher concentrations of the antibiotic (Figure 1-2-2-A). We hypothesized that mutants
defective in DST-mediated mRNA degradation would have increased abundance of the
HPH-DST transcript and would therefore have increased resistance to hygromycin relative
to the parental line. A preconstruction of the mutant selection revealed that seedlings
expressing the non-destabilized HPH message could be easily identified among a lawn of
seedlings expressing the destabilized HPH transcript (Figure 1-2-2-B). Because we were
interested in mutations affecting the DST pathway in trans, our goal was to identify mutants
with increases in both HPH-DST and GUS-DST mRNA abundance.

p1519-31 seeds were subjected to ethylmethane sulfonate (EMS) mutagenesis, sown
on soil and the M1 plants were allowed to self-fertilize (Figure 1-2-2-C)(16). M2 seeds were
collected from groups of MI plants and were plated on seed selection media containing 1000
rig/ml hygromycin (17). Out of ~730,000 M2 seeds that were plated, 323 seedlings that
appeared to exhibit increased resistance to hygromycin were transferred to soil for
propagation. By plating M3 progeny from these plants on selective media, we found that
approximately 20% of the original putative mutants displayed heritable hygromycin
resistance. In most of these lines, resistance was not attributed to an increase in HPH-DST
mRNA abundance. Three mutants, selected from independent M1 groups, Showed a
heritable increase in HPH-DST mRNA abundance; these three also Showed an increase in
G US-DS T mRNA abundance. Two of these mutants, dstl and dst2 that were isolated first,

have been characterized in greater detail.

85

A .-. ‘- C EMS mutagenesis of p1519-31
1000 r. ' * to generaie M1 seed
730,000 M2 seeds

plated on liygomycin

323 M2 seedlings
rescued

500

hygromycin (pg/ml)
00
O
O

 

20% heritable hygromycin
B resistance

1

Northern Blot Analysis

2 lines (dst1 and dsQ) with
heritable increases in
HPH-DST and GUS-DSTmRNA

 

Figure 1-2-2. The dst selection strategy. (A) A titration of hygromycin resistance was
performed on p1493-2 and p1519-31 seedlings. Approximately 500 seeds were surface
sterilized and plated on primary seed selection media containing 500, 800, or 1000 rig/ml
hygromycin (17). (B) In order to preconstruct the mutant selection, five p1493-2 seeds
were mixed with 500 p1519-31 seeds and plated on primary seed selection media
containing 1000 11ng hygromycin. All five resistant seedlings were recovered, one of
which is shown. (C) Steps in the dst selection.

The abundance of the HPH-DST and GUS-DST transcripts was 3 to 5 fold higher in
dstl and dst2 compared to p1519-31 plants (Figure 1-2-3-A). In contrast, the abundance of
the eIF4-A transcript, used as a non DST-containing control, was equivalent in mutants and
p1519-31 (Figure 1-2-3-B). In addition, two other mRNAs, encoding potential messenger
ribonucleases, that do not contain DST elements were equally abundant in p1519-31 and the
mutants (13). Increased HPH-DST and GUS-DST mRNA abundance is a consistent

phenotype that has been observed in the progeny of all backcrosses to p1519-31 (13).

86

Because both the HPH-DST and GUS-DS T transcripts were elevated in these mutants, it was

highly unlikely that the phenotype was due to a mutation in one or more of the DST

elements present in the 3’UTR of the HPH transcript. To examine this possibility directly,

the DST tetramer was amplified by polymerase chain reaction from genomic DNA extracted

from dst] and dst2 and was sequenced (1 8). The DST tetramers in both mutants were found

to be unaltered.

p1519-31

 

v- N
S ’4?
2 ”-dHPH-DST

T‘I

-‘ 4 GUS-DST

... ......- 4 eIF4-A

{

p1519-31
dst 1
dst 2

4 SAUR-AC1

any ,
.11]. '
.' ”1' '

m from" tows ‘ 9’F4'A

Figure 1-2-3. mRNA abundance of selected
transcripts in dst] and dst2. Total RNA was
prepared from leaves of 60-day-old plants and
analyzed by Northern blot hybridization (29). dst]
and dst2 plants derived from the first backcross to
p1519-31 were used as the source of material in this
experiment. Radiolabeled probes were prepared
against the indicated transcripts and were used in
sequential hybridizations of the same Northern blot.
(A) The abundance of DST-containing reporter
transcripts, HPH-DST and GUS-DST, are elevated
in dst] and dstZ. The higher molecular weight GUS
transcript (0) is a consistent characteristic of p15 1 9-
31. It is most likely the result of a partial recognition
of a cryptic downstream polyadenylation signal that
is activated by incomplete integration of the T-DNA.
We have measured the abundance of the lower
molecular weight band (the expected size) throughout
these experiments, although the two bands are
coordinately regulated in the mutants. (B) The
abundance of an endogenous DST-containing
message, SA UR-A C1, is elevated in dst] and dst2,
whereas the abundance of the eIF 4A (27) transcript,
which does not contain a DST element, is equivalent
in the mutants and p1519-31.

Genetic analysis indicated that dst] and dst2 are partially dominant mutations in

independent single genes. To arrive at this conclusion, we examined the inheritance of the

dst mutations by measuring HPH-DS T mRNA abundance in the rosette leaves of mature

87

plants derived fi'om crosses of dst] and dst2 to p1519-31 by Northern blot hybridization.
We used Northern blots rather than relying on reporter protein activity because direct
measurements were much more sensitive to the relatively small differences in mRNA
abundance observed in the mutants. Progeny of the second backcross to p1519-31 (F.) and
the progeny of self-fertilizations of these plants (F2) were used in these studies. Ten F.
plants were examined for each mutant. HPH-DST mRN A abundance was on average 2.1 i
0.22 fold higher in dst] F. and 2.8 i 0.23 fold higher in dst2 F. plants compared to p1519-
31 (19). These levels of HPH-DST mRNA abundance are intermediate between p1519-31
and mutant levels as would be expected if heterozygotes Showed a partial mutant phenotype.
Segregation of increased HPH-DST mRNA abundance in the F2 populations was also
consistent with partial dominance. Of 46 F2 plants segregating dst], nine were observed
with high HPH-DST mRNA abundance (3.1 i 0.17 fold p1519-31), 23 had intermediate
HPH-DST mRNA abundance (1.7 i 0.07) and 14 showed HPH-DST mRNA abundance that
was similar to pl 519-31 (0.9 i 0.04). Segregation of the dstZ mutant phenotype was similar
to that of dst]. Of 53 F2 plants analyzed, 13 fell into the mutant class (4.0 i 0.16), 28 fell
into the intermediate class (2.2 i 0.1 1), and 12 were similar to p1519-31 (1.0 i 0.04).
Segregation in dst] and dstZ F2 populations was most easily explained by a ratio of 1:2:1
(wild-type(wt):intermediatezmutant) as would be expected for inheritance of a partially
dominant Single gene (dstl x2: 1.1; dst2 x2: 0.2; p > 0.5).

Crossing dst] with dst2 yielded F. plants with intermediate HPH-DST mRNA
abundance, indicating that dst] and dst2 are not allelic. When the F2 plants from this cross
were analyzed, 10/51 showed HPH-DST mRNA abundance similar to p1519-31. Had the

dst mutations been in the same gene, or if they were tightly linked, we would not have

88

expected to observe any F2 plants with HPH-DST mRNA abundance similar to p1519-31.
No class of F2 plants was observed with an additive effect on HPH-DST mRN A abundance,
as might be expected if dst] and dst2 function independently to destabilize DST-containing
messages (20). In addition, the finding that F. plants from the dst] x dst2 cross showed an
intermediate mRNA abundance phenotype indicated a lack of additive interaction between
these loci because the individual mutants crossed to p1519-31 showed the same phenotype
in the F..

In order to begin the characterization of the molecular basis of these mutant
phenotypes, the dst] locus was mapped using molecular markers. This analysis showed that
dst] is located near position 68 on chromosome 5 (14).

If dst] and dst2 are indeed mutations that disrupt the DST-mediated mRN A
degradation pathway in trans, one would expect the abundance of endogenous DST-
containing messages to be elevated as well. We analyzed the abundance of the SA UR-A C1
transcript, the only DST-containing message in Arabidopsis that has been exarrrined at the
level of mRNA stability (3). The abundance of this message is elevated in dst] and dst2,
indicating that these mutations may elevate the abundance of any message destabilized by
DST sequences (Figure l-2-3-B). The abundance of messages that lack DST sequences was
unchanged in these mutants; for example, the abundance of the eIF4-A transcript is
equivalent in the mutants and p1519-31 (Figure 1-2-3-B). In addition, initial DNA
microarray experiments indicated, as expected, that most transcripts were not elevated in
dstI compared to p1519-31; however, a new SA UR mRNA, containing a potential DST

element, was among those that were more abundant in dst] (21). Taken together, these

89

observations suggest that the dst mutations Specifically affect the abundance of transcripts
containing DST instability sequences.

The Simplest explanation for the elevation of DST-containing transcripts in dst]
and dst2 is a defect that results in a decreased rate of DST-mediated mRNA degradation.
This is likely because the DST sequence is the only regulatory element Shared by the four
transcripts that were elevated in the mutants. To test this hypothesis, we measured the
kinetics of HPH-DST mRNA degradation in p1519-31, dst], and dst2 plants. We
anticipated that the measurable differences in HPH-DST mRNA decay rates would be
minimal because the difference in HPH-DST abundance between dst] or dst2 and p1519-
31 was only about three fold (Figure 1-2-3—A) and such differences are ofien dampened
by time course analysis using general transcription inhibitors, as discussed above (15).
Figure 1-2-4-A shows representative Northern blots from these experiments with the
average, normalized HPH-DST mRNA abundance at each time point plotted on a
semi-log scale in Figure 1-2-4-B. The average half-life of the HPH-DST transcript was
found to be 24 minutes for dst], 23 minutes for dst2, and 18 minutes for p1519-31
(Figure 1-2-4-B). The differences between mutant and p1519-31 mRN A decay curves
are particularly evident at later time points (Figure 1-2—4-B). We conclude that dst] and
dst2 cause an increase in message stability that results in increased abundance of DST-

containing mRNAs.

90

0’ 15’ 30’ 45’ 60’

 

 

 

A B
” “ p1519-31 g’
-: . .E
'2
g 0 p1519-31 (t1,2 = 18’)
“ m ’87" 03.21 at $2! dsa E U d3t1 (“,2 = 24’)
I t A dst2(t1,2 = 23’)
0.01 - . . .
0 15 30 45 60
time (min)

Figure 1-2-4. Analysis of HPH-DST mRNA stability in leaves from p1519-31, dst], and
dst2 plants. Rosette leaves from 12 F3 plants derived from the second backcross of dst]
and dst2 to p1519-31 were harvested. p1519-31 rosette leaves were harvested from 12
plants. Leaves were incubated in buffer for 30 minutes before addition of 150 rig/ml
cordycepin (30). Four leaves were harvested every 15 minutes for 60 minutes and frozen
in liquid nitrogen. Total RNA was extracted from each sample and analyzed by Northern
blot hybridization (29). Blots were hybridized with a radiolabeled probe complementary
to the HPH-DS T transcript and radioactive signals were quantititated by Phosphorlmager
(Molecular Dynamics). HPH-DST Si gnals were normalized by rehybridizing the blots
with a probe complementary to the eIF 4A transcript (27). (A) Representative Northern
blots Showing the HPH-DST signal over the 60-minute time-course in p1519-31, dst],
and dst2. (B) Average normalized HPH-DST signals (i standard error of the mean) from
the three time-courses are plotted on a semi-log scale. The average half-life of the HPH-
DST transcript is given on the graph. Results are expressed as the relative amount of
HPH-DST mRNA remaining at each time point after normalization with the eIF 4A Si gnal
compared to the first time point.

Neither of the dst mutants had any obvious defects in development or morphology.
Because expression of an auxin responsive gene, SA UR-A C], was altered in these mutants,
we analyzed phenotypes associated with this hormone. dst] and dst2 displayed normal root

gravitropism as indicated by the wavy root assay or by rotating vertical plates following

germination of seedlings (22). Additionally, there was no loss or enhancement of apical

91

dominance in these plants, nor were there alterations in root length or morphology, other
phenotypes that have been associated with auxin response mutants (23). The SA UR gene
family is large in Arabidopsis and, interestingly, there are several family members that are
highly similar in the coding region to SA UR-A C 1 yet lack DST elements (24). Therefore, it
is not surprising that a modest increase in mRN A abundance of one or a few of these genes
does not cause any obvious altered phenotypes. Although the number of endogenous
targets of the DST-mediated decay pathway is unknown, the dst mutants should be
powerful tools to address this question. For example, DNA microarray analysis Should
allow additional targets of the pathway to be identified by revealing mRNAs that
accumulate preferentially in either dst] or dst2.

The dst mutant selection had two interesting characteristics. First, these mutants
were extremely rare having been isolated at a frequency of less than l/200,000. In
Arabidopsis, null mutations in nonessential genes are typically expected at frequencies of
~1/2000 in EMS populations (25). Second, the dst mutations did not fully restore the
abundance of the HPH or GUS transcripts to the levels found in plants with non-destabilized
transcripts. These observations indicate that dst] and dst2 may be relatively weak alleles
that allow partial function of the DST-mediated mRNA decay pathway. If proper regulation
of DST-containing mRNAs is an essential function for Arabidopsis then this may have
precluded the isolation of stronger alleles. Altematively, functional redundancy among
components that recognize instability determinants may also contribute to the low
frequency of sequence-specific decay mutants. Perhaps it is by necessity rather than
coincidence that both dst mutants are partially dominant. Whether the corresponding

genes encode DST-binding proteins, DST-specific ribonucleases or other effectors,

92

dominant negative interactions between mutant derivatives and redundant proteins or
other components of the decay machinery are easy to imagine. Similar obstacles may
explain why successful selections for sequence-specific mRNA decay mutants have yet
to be reported in other eukaryotic systems.

Based on our work, selections for mutants defective in rapid mRNA degradation
mediated by Specific sequence elements should be feasible in other eukaryotic model
organisms if selectable marker genes that facilitate detection of small expression
differences can be engineered. The potential impact of this approach is demonstrated by
a series of very informative mutants of Saccharomyces cerevisiae and Caenorhabditis
elegans that are defective in a general mRNA decay pathway responsible for the
degradation of transcripts with premature stop codons (26). Finally, by providing the
means to clone dst genes, this work opens a new avenue to elucidate the molecular
machinery responsible for rapid sequence-Specific mRNA degradation in plants and will

likely provide novel information applicable to other eukaryotes.

93

REFERENCES AND NOTES

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2. T. C. Newman, M. Ohme—Takagi, C. B. Taylor, P. J. Green, Plant Cell 5, 701 (1993).
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10. Constructs were introduced into Arabidopsis using the vacuum infiltration method of
Agrobacterium tumefaciens mediated transformation [N. Bechtold, J. Ellis, G. Pelletier,
C.R.Acad.Sci.Paris 316, l 194 (1993) and Web site:
http://www.bch.msu.edu/pamgreen/vac.htm]. To avoid any hygromycin resistant
contamination of pl 5 19-31 during large-scale selection experiments, this construct was
introduced into a mutant that lacks trichomes (Col-PRL, glI). p1493 and other constructs
that confer hygromycin resistance were introduced into wt Columbia plants (Col-0).

11. L. Gritz and J. Davies, Gene 25, 179 (1983); R. A. Jefferson, T. A. Kavanagh, M. A.
Bevan, EMBOJ. 6, 3901 (1987).

12. M. L. Sullivan, D. M. Thompson, P. J. Green, unpublished data.
13. M. A. Johnson and P. J. Green, unpublished data.

14. Mapping of the p1519-31 transgene locus and the dst] locus were carried out using
SSLP markers [C. J. Bell and J. R. Ecker, Genomics 19, 137 (1994) and at the Web Site

94

http://genome.bio.upenn.edu/SSLP_info/SSLP.html]. F3 families derived from a cross
between p1519-31 and Landsberg erecta (glI-I) [M. Koomeef, L.W. Dellaert, J .H. van
der Veen, Mutation Research/, 93, 109 (1982)] were scored for kanamycin resistance. 15
families were identified that showed 100% kanamycin resistance. These were used to
map the transgene locus and were scored for 7 markers Spread across the genome.
Linkage was detected only to markers on chromosome 11, the closest marker being

ngal 126 (position 5 l , 0 recombinants out of 30 chromosomes analyzed). To map the
dst] locus, leaves of F2 plants from a cross of a'stI with Landsberg erecta (glI-I) were
scored for 3 fold or higher HPH-DST mRNA abundance compared to p1519-31 by
Northern blot analysis (19). 30 F2 plants were identified and scored for 18 markers
spread across the genome. Linkage was again detected with nga1126 (0 recombinants
out of 16 chromosomes analyzed) due to the necessity of the p1519 transgene for scoring
the mutant phenotype. In addition, linkage was detected to nga139 (map position 51, 2
recombinants out of 56 chromosomes analyzed) and nga76 (map position 68, 0
recombinants out of 56 chromosomes analyzed), defining the location of dst] on
chromosome 5.

15. A.-B. Shyu, M. E. Greenberg, J. G. Belasco, Genes Dev. 3, 60 (1989); J. G. Belasco
and G. Brawerman, in Control of messenger RNA stability, J. Belasco and G. Brawerman,
Eds. (Academic Press, San Diego,1993), ch. 18; S. Zhang, J. Sheng, Y. Liu, M. C.
Mehdy, Plant Cell 5, 1089 (1993); G. Caponigro and R. Parker, Microbiol.Rev. 60, 233
(1996); H. Holtorf, H. Sch6b, C. Kunz, R. Waldvogel, F. Meins, Jr., Plant Cell 11, 471
(1999)

16. Seeds homozygous at the p1519-31 locus (fourth generation of self-fertilized
progeny following transformation) were soaked for 16 hours in a 0.3% (v/v, water)
solution of EMS. Following mutagenesis, seeds were washed extensively with water
over a 12 hour period and then sown on soil. The effectiveness of EMS mutagenesis was
evaluated by scoring the occurrence of albinism and embryo lethality in M. plants as
described in G. P. Redei and C. Koncz, in Methods in Arabidopsis Research, C. Koncz,
N.-H. Chua, J. Shell, Eds. (World Scientific Publishing, Singapore, 1992) pp.16-82.

l7. Seed selection media consisted of 4.3 g/ L Murashige and Skoog salt mixture
(GibcoBRL), BS vitamin mixture (100 mg/l myo-inositol, 10 mg/l thiamine
hydrochloride, 1 mg/l nicotinic acid, 1 mg/l pyriodoxine), 1% sucrose, 0.5 g/L MES at
pH 5.7, 0.8% phytagar, 1000pg/m1 hygromycin and 50 ug/ml kanamycin.

l8. Genomic DNA was prepared from dst] and dst2 F2 plants from the first backcross to
p1519-31 as described in M. A. Saghai-Maroof, K. M. Soliman, R. A. Jorgensen, R. W.
Allard, Proc. Natl. Acad. Sci. 81, 8014 (1984). PCR primers complementary to the 3’ end
of the HPH coding region and the 5’ end of the E9 3’UTR were used to amplify the DST
elements with a polymerase with proof-reading capability (Pfu, Stratagene) under
standard PCR cycling conditions. Products were ligated into a derivative of pBluescript
SKII(-) and multiple individual clones were sequenced for each reaction.

95

19. Signals from Northern blots were quantitated with a Phosphorlmager (Molecular
Dynamics). The HPH-DS T Signal was normalized using the elF 4A (27 ) signal. p1519-
31 samples were analyzed in parallel and the average fold increase of segregants
compared to p1519-31 HPH-DST mRNA abundance is reported i the standard error of
the mean. RNA was extracted from two rosette leaves of individual plants as described
by T. C. Verwoerd, B. M. Dekker, A. Hoekema, Nucleic Acids Res 17, 2362 (1989).
Northern blots were processed as described previously (2).

20. In the F2 of a cross between two partially dominant mutations with no additive effect
on one another, such as is proposed here, one would expect a ratio of 1:8:7
(wt:Intermediate:Mutant) or 12826 if double mutants are not viable. We observed 10 wt,
26 intermediate, and 15 mutant plants out of 51 F2 plants that were tested. This gives a
ratio of ~3:8:5, which does not precisely fit simple models of inheritance. Because it is
clear that dst] and dst2 are partially dominant mutations in single genes, this may
indicate subtle interactions between these loci. For example, dst! and dst2 may partially
suppress one another, explaining the higher than expected proportion of wt segregants.

21. M. A. Perez-Amador, M. A. Johnson, J. Landgraf, E. Wisman, P. J. Green,
unpublished data.

22. C. J. Bell and E. P. Maher, MGG 220, 289 (1990); K. Okada and Y. Shimura,
Aust.J.Plant Physiol. 19, 439 (1992).

23. W. Boerjan et al., Plant Cell 7, 1405 (1995); L. Hobbie and M. Estelle, Plant Cell
Environ. 17, 525 (1994); H. M. O. Leyser, F. B. Pickett, S. Dharrnasiri, M. Estelle, Plant
J. 10, 403 (1996).

24. T. J. Guilfoyle, personal communication; M. A. Johnson and P. J. Green, unpublished
sequence database search results.

25. G. Haughn and C. R. Somerville, in Agricultural Chemistry, H. M. LeBaron, R. O.
Mumma, R. C. Honeycutt, J. H. Duesing, Eds. (American Chemical Society,
Washington, DC, 1987) p. 98.

26. P. Leeds, S. W. Peltz, A. Jacobson, M. R. Culbertson, Genes Dev. 5, 2303 (1991); R.
Pulak and P. Anderson, Genes Dev. 7, 1885 (1993); Y. Cui, K. W. Hagan, S. Zhang, S.
W. Peltz, Genes Dev. 9, 423 (1995); F. He and A. Jacobson, Genes Dev. 9, 437 (1995);
B. S. Lee and M. R. Culbertson, Proc.Natl.Acad.Sci. USA 92, 10354 (1995); B. M. Cali,
S. L. Kuchma, J. Latham, P. Anderson, Genetics 151, 605 (1999).

27. The eukaryotic translation initiation factor 4A (eIF4-A) gene was cloned from
Arabidopsis by C. B. Taylor, P. A. Bariola, S. B. DelCardayre', R. T. Raines, P. J. Green,
Proc.Natl.Acad.Sci. USA 90, 51 18 (1993).

28. p1519 and p1493 are derivatives of pMON505-70 [R.-X. Fang, F. Nagy, S.
Sivasubramaniam, N.-H. Chua, Plant Cell 1, 141 (1989)]. The HPH coding region was
obtained as a BamHI fragment of pLG90 [L. Gritz and J. Davies, Gene 25, 179 (1983)].

96

Use of the 35S promoter, polyadenylation Signals from the 3’ ends of the E9 and 3C
genes, and synthesis and insertion of the DST tetramer were as described previously (2).

29. RNA extraction and Northern blot analysis was performed essentially as described
by T. C. Newman, M. Ohme-Takagi, C. B. Taylor, P. J. Green, Plant Cell 5, 701 (1993).

30. Cordycepin (150 ug/ml) was used to inhibit transcription in mature leaves because
actinomycinD was less effective in this tissue (13). This protocol, including the leaf
incubation medium (lmM Pipes, pH 6.26; lmM sodium citrate; lmM KCl; 15 mM
sucrose) was adapted from previous reports [K. A. Seeley, D. H. Byrne, J. T. Colbert,
Plant Cell 4, 29 (1992); H. Holtorf, H. Schéb, C. Kunz, R. Waldvogel, F. Meins, Jr.,
Plant Cell 11, 471 (1999)].

31. We thank Dr. Ambro van Hoof, Dr. Michael Thomashow and James Kastenmayer
for reading the manuscript and Dr. Joanne Chory for providing pLG90. We also thank
Dr. Michael Sullivan and Debrah Thompson for construction of pl 519 and p1493; and
Jonathan Vogel and Linda Danhof for technical assistance. This work was funded by
grants from the United States Department of Energy, the United States Department of
Agriculture, and the McKnight Foundation to P.J.G. M.A. P-A. received postdoctoral
fellowships from NATO-Spain and from the Ministerio de Educacion y Ciencia, Spain.

97

CHAPTER 1-3

ANALYSIS OF THE DST ELEMENT AND DST-LIKE SUBDOMAIN S

WITHIN THE SA UR-A C1 3’ UNTRANSLATED REGION

98

INTRODUCTION

The DST element is perhaps the best-studied instability determinant that is plant-
specific. The demonstration that this sequence is sufficient to destabilize messenger
RNA (mRNA) came when Newman et al. showed that a dimer of the element, when
inserted into the 3’ untranslated region (UTR), targeted otherwise stable reporter RNAS
for rapid turnover (1993). The DST element was originally found in the 3’ UTR of the
unstable small-auxin-up-RNA (SA UR) genes and has been found in the 3’UTR of several
SA URs from diverse plant Species (McClure et al., 1989; Yamamoto et al., 1992; Gil et
al., 1994). The element consists of three conserved subdomains separated by two
variable regions. These subdomains are referred to as GGA, ATAGAT, and GTA after
the DNA sequence that comprises the core of each conserved region. Sullivan and Green
showed that substitution mutations in either the ATAGAT or GTA subdomains abolished
the effect of the DST dimer (1996), demonstrating the necessity of these sequences for
DST function.

The experiments described above explored the firnction of a synthetic DST
sequence derived from the soybean SA UR-15A gene (McClure et al., 1989). In order to
study a DST element in its native context, Gil and Green cloned the SA UR-A C1 gene
from Arabidopsis (Gil et al, 1994). The 3’UTR of this gene is sufficient to target reporter
transcripts for rapid turnover, reducing the half-life of the human ,B-globin (Globin)
transcript to a Similar extent as did a dimer of the DST element (Newman et al., 1993; Gil
and Green, 1996). While these two experiments are not directly comparable because the

half-life measurements were done using different methods, this result raises some

99

interesting questions because the SA UR-A C1 3 ’ UTR contains only one canonical DST
element. Previous experiments indicated that a monomer of the DST element was not
sufficient to cause rapid turnover of reporter transcripts (Newman et al., 1993) and
experiments reported in Chapter 1-1 of this thesis indicate that a DST tetramer is more
effective as an instability determinant than a dimer. These results would support the
hypothesis that DST elements serve as recognition sites for the mRNA degradation
machinery and become more and more effective as additional copies are added, with a
minimum of two copies required for function. The function of the SA UR-A CI 3’UTR as
an instability determinant, which contains one DST element, would appear to contradict
this assertion. To test the contribution of the DST element to SA UR-A C1 3’UTR
instability function, Gil and Green made substitution mutations in the ATAGAT and
GTA subdomains (unpublished data). These mutations, tested individually, reduced the
abundance of reporter transcripts to the same extent as did the wild type SA UR-A C1
3’UTR, indicating that individually, they are not required for SA UR-A C1 3’UTR
instability function. Interestingly, upon analysis of the sequence of the SA UR-A C1
3’UTR upstream of the DST element, several potentially redundant ATAGAT- and GTA-
like subdomains were noted. This raised the possibility that perhaps these redundant
elements were contributing to the instability fiinction of the SA UR-A C1 3’UTR and were
providing the additional DST sequences that experiments with synthetic elements had
Shown to be necessary.

The purpose of the experiments outlined in this chapter was to analyze the
contribution of these ATAGAT and GTA-like subdomains and to enhance our

understanding of DST function by studying the element in its native context. Two

100

approaches were taken. First, all DST and DST-like subdomains were replaced with the
same substitutions that had been shown to abolish the function of the DST dimer
(Sullivan and Green, 1996). In the second approach, the SA UR-ACI 3’UTR was divided
into three regions and each was tested for instability function. Both of these approaches
were somewhat risky because the DST element overlaps polyadenylation Signals within
the SA UR-A C I 3 ’UTR. We expected that chimeric transcripts might be aberrantly
polyadenylated, however, because so little is known about polyadenylation signals in
plants, there was no way to know for certain until we analyzed the constructs in
transgenic plants. In some cases, this limitation has made the results of these experiments
difficult to interpret. However, this study suggests a possible role for upstream redundant
ATAGAT and GTA-like subdomains in the instability function of the SA UR-ACI 3’ UTR
and provides some interesting tools for future analysis of DST-mediated mRNA

degradation.

RESULTS

Mutagenesis of all DST and DST-like subdomains

The 3’UTR of the SA UR-A C1 transcript contains one DST element along with
other DST-like subdomains that are iterated upstream. In Figure 1-3-1, the DST element,
ATAGAT-, and GTA-like subdomains have been highlighted. The ATAGAT subdomain

consists of the sequence ATAGAT and the GTA subdomain consists of the sequence

101

CAATGCQE, ATAGAT-like subdomains were defined as having one mismatch and
GTA-like subdomains were defined as having one or two mismatches.

In order to assess the contribution of the DST element and ATAGAT and GTA-
like subdomains to SA UR-A C1 instability function, all of the residues that are highlighted
in Figure 1-3-1 were mutated. ATAGAT and ATAGAT-like subdomains were replaced
with GCATGC, changing 5 of the Six conserved residues. GTA subdomains
(CAATGCGTA) were replaced with CATAGGCCT which changes the GTA residues,
conserved in all SA UR DST elements, and three residues upstream. Either of these
mutations iS known to abolish the function of the synthetic DST dimer (Sullivan and

Green, 1996).

 

I!

 

AGTAC TATA CTACAACATTTC CA TA'ITT'ITI'ITAG AéI'I'G TTA

 

 

GCTAATTTC cc crc. GAGATAA'lTG TAAATTGTITCAATG AG

 

 

 

I

:54»: '45:?
. DST element
TTGCATGTTAAAAA '~' ~ - r"
...m..- GTA-like sequence

mew-«3.4:.» ATAGAT-like sequence

Figure 1-3-1. The SA UR-A C1 3’UTR contains one DST-element and DST-like
subdomains. The sequence of the SA UR-A C1 3’UTR immediately 3’ of the stop codon to
the beginning of the poly(A) tail is shown (Gil et al., 1994). The DST subdomains
(“GGA”, “ATAGAT”, and “GTA”, are highlighted by gray boxes. DST-like subdomains
are highlighted by bars and arrows as described in the figure.

The fully mutated SA UR-A C1 3’ UTR was constructed by polymerase chain
reaction using four overlapping oligonucleotides. These mutations altered 29% of the

nucleotides in the SA UR-A C1 3’UTR, changing the GC content from 29% to 45%. The

fully mutated 3 ’UTR was inserted 3 ’ of the Globin coding sequence and the resulting

102

chimeric gene was introduced into a binary vector containing a GUS reference gene so
that the effect on message stability could be assessed in transgenic plants (Figure 1-3-2).

Control constructs containing the wild type SA UR-A C I 3’UTR and the E9 3’UTR, were

also generated.

 

 

-[Es>| Globin I 59]
, T t
@I Globin [SAUR] es

 

 

 

 

Reference

 

 

Figure 1-3-2. The structure of chimeric genes used to test mutations in the SA UR-ACI
3’UTR. The cauliflower mosaic virus 35$ promoter (35S) was used to control the
transcription of test and reference genes. The reference gene, B-glucuronidase (GUS),
was included in the same binary vector as the test gene to facilitate normalization of
mRNA abundance data. The 3 ’UTRS of the pea ribulose 1,5-bisphosphate carboxylase
small subunit E9 (E9) and 3C (3C) genes were used as a negative control (lacking
instability activity) and as a 3’UTR for the reference gene, respectively. SA UR represents
the wild type SA UR-A C1 3’UTR, while saur (shaded) represents the fully mutated
version. The right border (RB) of the transfer-DNA (T-DNA) has been indicated by a
black rectangle. The T-DNA also includes the neomycin phosphotransferase cassette so
that kanamycin resistant transgenic plants could be selected.

These constructs were introduced into Arabidopsis via the vacuum infiltration
method of Agrobacterium tumefaciens mediated transformation (Bechtold et al., 1994).
Approximately 100 independent, 12-day-old primary transforrnants (T.) from each

construct were pooled and harvested for Northern blot analysis (Figure 1-3-3).

103

Figure 1-3-3. Mutations in the SA UR-ACI 3’ UTR
affect polyadenylation of the Globin reporter
transcript. A Northern blot containing 10 pg of
total RNA from pools of T. seedlings was
he < Globin hybridized sequentially with radiolabelled Globin

' A i and GUS probes. The 3’UTR of each Globin
1 2 3 reporter is given above each lane (E9, wild type

SA UR-A C1, fully mutated SA UR-A C1).

MMM <GUS

E9
SAUR
saur

The top panel of this Northern blot Shows the abundance of Globin transcripts
containing the three different 3’UTRS that were tested. The Globin-SA UR transcript was
2.1 fold less abundant than the Globin-E9 transcript after each signal was normalized
with the reference Si gnal (GUS) (compare lanes 1 and 2). This difference was less than
that observed by Gil and Green (4 fold, 1996), but still indicates that the wild type SA UR-
AC1 3’UTR functioned as an instability determinant. As one can see on examination of
lane 3, the fully mutated SA UR—A CI 3’UTR results in larger Globin transcripts. This was
most likely due to the inadvertent mutation of polyadenylation Signals within the SA UR-
ACI 3’UTR. The larger transcripts were probably produced when downstream
polyadenylation sites within the T-DNA were utilized, this will be discussed further
below.

T. plants were allowed to self-fertilize and the progeny were collected so that T2
plants could be analyzed. Approximately 100 T2 seedlings from 10 independent
transformants were plated and harvested for Northern blot analysis. AS expected, the
results with these pools of seedlings were similar to those Shown in Figure 1-3-3 (data not
shown). Because aberrant transcripts were produced from these constructs, it was not

possible to address the contribution of DST and DST-like subdomains to the

104

accumulation of the Globin transcript using this approach. In order to overcome this
obstacle, a second approach was undertaken in which the SA UR-A C1 3’UTR was divided

into sections and each was tested for instability function.

Gain of function analysis of DST and DST-like subdomains

Another way to test the contribution of the DST-like subdomains present in the 3’
UTR of SA UR-A C] was to test whether they have stand-alone instability function. This
approach has been used successfully in our laboratotory to Show that the DST element
and AUUUA repeats are instability determinants. One simply inserts the sequence to be
tested between the coding region of a reporter gene and a 3’UTR that provides
polyadenylation signals. The SA UR-A CI 3’UTR was divided into three sections that
contained go DST-like subdomain (N), redundant DST-like subdomains (R), and the
DST element (D). The N region consisted of the first 31 nucleotides of the SA UR-A C1
3’UTR, ending at the first arrow shown in Figure 1-3-1. The R region was defined as the
next 48 residues, containing one ATAGAT-like subdomain and three GTA-like
subdomains, ending five nucleotides upstream of the boxed DST element shown in
Figure 1-3-1. The D region comprised the next 52 nucleotides and ended 8 nucleotides
downstream of the GTA subdomain Shown in Figure 1-3-1. Sections of the 3’UTR were
constructed using overlapping oligonucleotides and were inserted between the Globin
coding region and the E9 polyadenylation signal, as depicted in Figure 1-3-4. The same
test and reference gene strategy described above was used to construct binary vectors to

be introduced into Arabidopsis.

105

 

A. I SAUR-AC1 INI RI DJ

B. @flflobm INI E9I
e e T t
-I:3_?_S>I Globin I a has I es
-Es>rclobinl o I ab

E355>I GUS I acJ-m Reference

Figure 1-3-4. Structure of chimeric genes used to test individual subdomains within the
SA UR-A C1 3’UTR for instability function. A. The SA UR-A C1 gene structure is depicted
with the 3’UTR divided into sections called N, R, and D as described in the text. B.

Constructs used in this study were derivatives of those described in Figure 1-3-2.

 

 

 

 

 

 

 

 

Transgenic plants expressing these constructs were tested for instability function
using the same method described above. Figure 1-3-5 Shows the results of Northern blot
analysis of RNA isolated from pools of T. seedlings. Pools of seedlings expressing the
Globin reporter with the E9 3’UTR (Globin-E9) alone were included in the analysis as a
negative control. Insertion of the N, R, and D regions resulted in an increase of the
Globin transcript size that was expected for each (Figure 1-3-5-A). By comparing the
ratios of Globin/ GUS mRNA accumulation presented in Figure 1-3-5-B, it was found that
the N region, had almost no effect on message accumulation in transgenic seedlings. In
contrast, the D region had a mild effect (71% of E9) and the R region had a large effect
on the accumulation of the Globin reporter mRNA, reducing the abundance to 37%

compared to plants expressing Globin with the E9 3’UTR alone.

106

A B 1.2-
3 3 3 (0 921, 37% 71% IBM“
I I I a
z 0: o 3 g 0.9- EIBlot2
I M “U <clobin s 06.
e o
0
.2
,, ,, 5
fifluu-WGUS £
‘

 

 

 

 

     

232

Figure 1-3-5. Analysis of the N, R, and D regions of the SA UR-A CI 3’ UTR. A. A
Northern blot containing 10 ug of total RNA from a pool of T. seedlings was hybridized
sequentially with radiolabeled Globin and GUS probes. The 3’UTR of each construct is
given above each lane. B. Globin and GUS transcript abundance was quantitated using a
Phosphorlmager. Globin transcript abundance was normalized using the GUS abundance
and these values were plotted relative to the E9 control. The values obtained fi'om
independent Northern blots are shown. The average Globin/GUS ratio from the two blots
is given above each bar and is expressed as a percentage of the Globin-E9 value.

These results pointed to a possible role for the R region in the instability function
of the SA UR-A C1 3’UTR. In addition, it is interesting to note that the D region was not
sufficient to reduce the abundance of the Globin reporter. This result is compatible with
those of previous experiments that showed that mutating the ATAGAT or GTA
subdomains within the DST element had no effect on the function of the SA UR-A C1
3’UTR as an instability determinant (P. Gil and P.J. Green, unpublished). These results
are also in agreement with the finding that a monomer of the DST element from the
soybean SA UR-15A gene is insufficient to destabilize Globin transcripts (Newman et al.,
1993). These observations indicated that perhaps the R and D regions of the SA UR-A C1
3 ’UTR function together to achieve full instability function. In order to test this

hypothesis, Globin constructs containing combinations of the N,R, and D (NR, RD, NN,

107

RR, and DD) regions were introduced into Arabidopsis. These constructs had the same
structure as those shown in Figure 1-3-4 and were analyzed in the same way, using pools
of T. seedlings.

The results of this experiment are presented in Figure 1-3-6. When the R region
was introduced into the Globin transcript as a dimer (RR) or in combination with the D
region (RD), smaller transcripts were produced. This is clear in lanes 2 and 4 of Figure
1-3-6-A. These smaller transcripts are most likely the result of cleavage and
polyadenylation at Sites within the introduced sequences. This conjecture is reasonable
considering that the SA UR-A C1 polyadenylation Signals are likely located within the N,
R, and D regions. The larger transcripts were thought to be full length and would
therefore contain the introduced putative instability sequences and sequences from the E9
3’UTR. In order to verify that the larger transcripts were full-length, the blot shown in
Figure 1-3-6-A was stripped and rehybridized with a probe complementary to the E9
3’UTR (Figure 1-3-6-B). Only the larger transcripts were detected with the E9 probe.

Polyadenylation within the introduced dimers of N, R, and D regions confounded
analysis of their impact on message stability based on mRNA abundance. The smaller
transcripts present in lanes 2 and 4 of Figure 1-3-6-A probably do not contain the entire
RD or R sequences and may therefore be relatively stable following cleavage and
polyadenylation. In addition, it is difficult to make conclusions about the instability
function of these sequences based solely on the abundance of the full-length-transcript
because the abundance of this species is dependent upon the rate of cleavage and

polyadenylation at Sites within the introduced sequences.

108

 

 

   

3.1

a a a a a c
a: d 2 r: d. a,
z a: z m o '” 1' IBlot1
. ,, _ nmmz
'3 ’2‘ ' 1 """ . ‘ GIObln o_3.
Mi ' u:
3
(5
E 06.54% 27% 40% 20% 52%
1 2 3 4 5 6 g'
G
unmunu<cus go...
3
0
‘F 0.2-
B
“I .. w 0...? m . ‘E9 0. 1
B B 81 B
0': d 2 0':
z I z I

3
d
a

Figure 1-3-6. Analysis of combinations of N, R, and D regions. A. A Northern blot
containing 10 ug of total RNA from pools of T. seedlings was hybridized sequentially
with radiolabeled Globin and GUS probes. The 3’UTR of each construct is given above
each lane. B. The blot was stripped and hybridized with a radiolabeled probe
complementary to the E9 3’UTR. C. Full-length Globin and GUS transcript abundance
was quantitated using a Phosphorlmager. Globin transcript abundance was normalized
using the GUS abundance and these values were plotted relative to the abundance of the
Globin-E9 control transcript. The values obtained from independent Northern blots are
shown. The average Globin/GUS ratio from the two blots is given above each bar and is
expressed as a percentage of the Globin-E9 value.

For the sake of discussion, the abundance of each firll-length Globin transcript
was quantitated and is plotted relative to the Globin-E9 transcript in Figure 1-3-6-C.
From these data, it would appear that the R region has a greater effect on Globin message
abundance when it is paired with the D (RD, 27% of E9) region then when paired with
the N region (NR, 54% of E9). However, this may simply reflect the obvious difference
in internal polyadenylation between these two constructs. Likewise, the RR dimer had

the largest impact on the abundance of the Globin message (28% of E9) compared to the

109

NN dimer (40% of E9) or the DD dimer (52% of E9). Again, these data must be
considered with caution because the RR dimer served as a site for internal cleavage and

polyadenylation.

DISCUSSION

The SA UR-A C1 3’UTR, which contains one DST element and several DST-like
subdomains, is a mRNA instability determinant that contributes to the constitutive
instability of the SA UR-A C1 message (Gil and Green, 1996). The experiments described
in this chapter were designed to assess the contribution of the DST element and DST-like
subdomains to the overall instability function of the SA UR-A C1 3’UTR. The goal of this
work was to gain a better understanding of how the DST element in particular, and
instability determinants in general, function within their native context. Two approaches
were taken, one in which all of the DST and DST-like subdomains were mutated and a
second where the SA UR-A C1 3’UTR was divided into three regions and each was tested
for instability function.

Mutating all of the DST and DST-like subdomains apparently abolished the
polyadenylation Signals within the SA UR-A C1 3’UTR and resulted in aberrant, large
reporter transcripts when expressed in transgenic plants (Figure 1-3-3). This made it
impossible to pursue this approach further. It was expected that these mutations would
have resulted in stabilization of the reporter transcript. The function of individual
subdomains was to be addressed by adding back wild-type sequences and analyzing the

effect on reporter transcript abundance. We were aware of the potential for problems due

llO

to incorrect polyadenylation of these chimeric transcripts, so we devised an alternative
strategy.

The second approach was more informative. In these experiments, the SA UR-
AC1 3’UTR was divided into three regions called N (no DST-like subdomain), R
(redundant DST-like subdomains), and D (DST element). These sequences were
introduced into the 3’UTR of a Globin-E9 reporter message and were studied using
Northern blot analysis of RNA extracted from transgenic Arabidopsis plants. These
experiments indicated that the R region may have instability function. The introduction
of this sequence into the Globin-E9 3’UTR caused an almost three-fold decrease in
message abundance compared to Globin-E9 (Figure 1-3-5). Interestingly, the R region
was more effective at reducing reporter transcript abundance than was the D region. The
instability function of a dimer of the DST element derived fi'om the soybean SA UR-15A
gene is well established (Newman et al., 1993; Sullivan and Green, 1996), however, a
monomer of the element is much less effective (Newman et al., 1993). Therefore, the
finding that a monomer of the D region of the SA UR-A C1 3’UTR did not greatly reduce
Globin mRNA abundance was perhaps not surprising. Nonetheless, these results raise
some intriguing questions: IS a dimer of the DST element necessary because recognition
factors that bind this sequence only function as dimers? Do the redundant DST-like
subdomains present within the R region of the SA UR-A C1 3’UTR serve as an alternative
binding site(s) for DST recognition factor(s)? Do the R and D regions function
synergistically to achieve instability function?

To begin to address these types of questions, homo- and heterodimers of the N, R,

and D regions were analyzed (Figure 1-3-6). One important question was whether RD

111

would reduce mRNA abundance of the Globin reporter to a greater extent then ND.
Unfortunately, when the RD region was introduced into the Globin-E9 3 ’UTR, two
transcripts were apparent on Northern blots hybridized with Globin probes. The simplest
explanation for the presence of the smaller band was that the RD sequence had served as
an internal polyadenylation signal. This conjecture was supported when only the larger
band hybridized to a probe complementary to the E9 3’UTR, located downstream of the
RD insertion. A smaller than expected transcript was also produced when a homodimer
of the R region was analyzed. This complication was unexpected because the 3’ end of
the SA UR-A C1 transcript has been identified and is known to be downstream of the N, R,
and D regions (Gil et al., 1994). When the R region was introduced into the Globin
3’UTR as a homodimer or as a heterodimer with the D region, unexpected
polyadenylation signals may have been created. Plant polyadenylation signals are poorly
defined and they generally do not contain the AAUAAA sequence that marks the
polyadenylation site in almost all mammalian transcripts (reviewed by Hunt, 1994).
Indeed, prediction of polyadenylation Si gnals within individual plant genes seems to be
nebulous at best. The lack of sophisticated knowledge of poly(A) Site selection in plants
makes it difficult to determine if the R region contains partial polyadenylation Signals
that may have been activated upon dimerization or when it was combined with the D
region.

In any case, this complexity precluded analysis of the RD and RR sequences in
terms of their instability function by Simply assaying mRNA accumulation. The data
obtained by analyzing the abundance of the full-length transcript were interesting because

they provided support for the hypothesis that the R and D regions function together. The

112

Globin/GUS ratio was lowest in transgenic plants with the RD and RR sequences inserted
into the Globin 3’UTR. However, conclusions cannot be drawn fiom these results
because the contribution of internal polyadenylation to the decreased accumulation of the
full-length transcript in these plants cannot be estimated.

A method that uses the general transcription inhibitor, cordycepin, to measure
mRNA stability in Arabidopsis plants has been developed and is described in Chapter 1-2
of this thesis. This method may now be used to directly measure the effect of the R
region on message stability using the transgenic plants generated for this study. The
demonstration that the R region results in rapid turnover of the Globin reporter would
solidify the hypothesis that this region plays an important role in rapid mRNA decay
mediated by the SA UR-A C1 3 ’UTR. In addition, if one assumes that transcripts detected
on Northern blots are largely mature, cytoplasmic mRNAs, then it may be possible to
address the effect of the RD and RR dimers on message stability by conducting half-life
analysis on the full-length transcript. These experiments would allow the stability of the
full-length transcripts to be addressed without regard for the steady-state abundance,
which is probably influenced by internal polyadenylation due to insertion of the RR and
RD sequences.

In Chapter 1-2 of this thesis, two mutants defective in DST-mediated mRN A
degradation were described (dstI and dst2). One of the interesting features of these
mutants was that they both had elevated SA UR-A CI message abundance. Interestingly,
this effect was greater in dst] compared to dst2. The abundance of the hygromycin
phosphotransferase (HPH) mRNA that was destabilized by a DST tetramer (HPH-DST)

was also elevated in both mutants, but is higher in dst2 (Figure 1-2-3). These subtle

113

differences in the phenotype of these mutants may point to differences in recognition of
independent DST and DST-like subdomains. For example, perhaps the dst] gene product
is involved in the recognition of GTA subdomains and the dst2 gene product is involved
in the recognition of ATAGAT subdomains. The tetramer of the DST element contains
an equal ratio of ATAGAT to GTA subdomains, the two subdomains important for DST
function (Sullivan and Green, 1996). The proportion of GTA-like subdomains is higher
in the SA UR-A C1 3 ’UTR, it contains four ATAGAT-like and five GTA-like subdomains
(Figure 1-3-1). Because the SA UR-A C1 3 ’ UTR has a higher proportion of GTA-like
subdomains and the dst] mutant shows hyperelevation of the SA UR-A C1 transcript
relative to dst2, one might speculate that the dst] gene product is involved in recognition
of GTA subdomains. Likewise, if the dstZ gene product was responsible for recognition
of the ATAGAT subdomain, one might expect the defect to be more obvious on the
HPH-DST message because it has a higher proportion of ATAGAT subdomains.

The R region, implicated in SA UR-A C1 function by this study, has one ATAGAT-
like subdomain and three GTA-like subdomains. If the above speculation is correct, one
might expect that the dst] mutant would Show hyperelevation of the GIobin-R-E9
reporter transcript relative to the dst2 mutant. Therefore, the Globin reporter transcripts
and transgenic plants generated for this study might allow us to learn more about the
function of the dst] and dst2 gene products by introducing these constructs into the
mutant backgrounds and determining whether the mutants differentially recognize
independent regions of the SA UR-A C1 3 ’UTR.

Future studies into DST-mediated mRNA degradation will be enhanced by

combining the reporter constructs described in this chapter with further analysis of the

114

mutants described in the previous chapter. It seems probable that recognition of synthetic
DST elements and those residing in native transcripts, such as SA UR-A CI, is a modular
process. It will be fascinating to uncover the cellular factors that comprise recognition
modules and to understand how they interact with the basal mRNA degradation

machinery to achieve rapid turn over of DST-containing messages.

MATERIALS AND METHODS

Plasmid construction and plant transformation

Standard molecular cloning procedures were used to construct the chimeric genes
described in Figure 1-3-2 and Figure 1-3-4 (Sambrook et al., 1989). The fully mutated
SA UR-A CI 3’UTR (Figure 1-3-2) was synthesized using four oligonucleotides. Two 98
base oligonucleotides that overlapped by 24 nucleotides were synthesized that
incorporated all of the mutations in DST and DST-like subdomains. These were
annealed and the overhanging ends were filled by 8 cycles of polymerase chain reaction
(PCR). The double stranded product of this PCR was then subjected to a second round of
PCR (25 cycles) using two smaller primers (24 and 26 bases) complementary to each
end. The oligonucleotides were designed such that the 5’ end of the final double-
stranded product was a BamHI site and the 3’ end was a KpnI site. This facilitated
introduction of the mutated portion of the SA UR-A C1 3’UTR into a version of the entire
SA UR-A CI 3’UTR that contained a BamHI Site four base pairs 3’ the stop codon and a

KpnI site 15 base pairs 3’ of the end of the SA UR-A C I transcript. Only one insertion and

115

one substitution were required to introduce these restriction sites using mutagenic
oligonucleotides and the Kunkel method of site-directed mutagenesis (Kunkel et al.,
1987). The fully mutated SA UR-A C1 3’UTR, and the SA UR-A C1 3’UTR with the BamHI
and Kpnl sites (wt) were introduced into a pUC derivative with the structure:
3SS:Globin:E9 (p1630) by replacing the E9 3’UTR with the fiilly mutated SA UR-A C1
3’UTR (pl658) or the wt SA UR-ACI 3’UTR (p1657). Three test genes 3SS:Globin:E9,
35S:Globin:wt SA UR (with BamHI and Kpnl Sites) and 35S:Globin:fully mutated SA UR
were used in this study. The 353 promoter in these three constructs was a Shorter version
(-417 to +8; Feldbrfigge et al., 1994) than that used by Newman et al., (1993) (—940 to
+9) which was used for all of the other constructs.

The N, R, D, NR, RD, NN, RR, and DD regions of the SA UR-A C1 3’UTR were
constructed by synthesizing overlapping, complementary oligonucleotides with a BglII
site on the 5’ end and a BamHI Site on the 3’end. Oligonucleotides were annealed and
introduced at the BamHI site of a pUC derivative containing a test gene with the
structure: SacI-35S-BglII-Xbal-Globin coding region-BamHI-E9-Clal (Newman et al.,
1993). Homodimers were constructed by screening for clones with two insertions of the
annealed oligonucleotides.

Test genes, described above, were transferred to a derivative of pMON505 which
is a binary vector that facilitates transformation of plants via Agrobacterium tumefaciens
(Agrobacterium) (Rogers et al., 1987, Fang et al., 1989). The transfer DNA also contains
a reference gene with the structure 3SS-GUS-3C. This test and reference strategy and the

source of the Globin and GUS coding regions was described by Newman et al., (1993).

116

Generation and selection of transgenic Arabidopsis plants using the vacuum
infiltration method of A grobacterium-mediated transformation has been described (N.

Bechtold et al., 1993 and Web site: http://www.bch.msu.edu/pamgreen/vac.htm).
Agrobacterium strain GV3101 CS8C1 Rif (pMP90) (Koncz and Schell, 1986) was

transformed with pMON505 derivatives by electroporation using the Gene-Pulser

(BioRad) according to the manufacturer’s guidelines.

Plant material and growth conditions

The Columbia (Col-O) accession of Arabidopsis thaliana was used in all
transformations. Arabidopsis seedlings were grown on seed selection media which
consisted of 4.3 g/ L Murashige and Skoog salt mixture (GibcoBRL), BS vitamin mixture
(100 mg/l myo-inositol, 10 mg/l thiamine hydrochloride, 1 mg/l nicotinic acid, 1 mg/l
pyriodoxine), 1% sucrose, 0.5 g/L MES at pH 5.7, 0.8% phytagar, and 50 ug/ml
kanamycin. Plates were kept in incubators set at 16 hours light (125 uE/mz) /8 hours
dark, 21°C. Arabidopsis plants were grown in standard Arabidopsis soil in controlled

environment growth chambers set at 16 hours light (125 uE/mz) /8 hours dark, 21°C.

RNA analysis

RNA was isolated from pooled seedlings using the method described by Puissant
and Houdebine (1990) with modifications described by Newman et al., (1993). T. pools

consisted of ~200 individual seedlings, all of which were likely to be from independent

117

transformation events (Ye et al., 1999). T2 pools consisted of ~100 individual seedlings
from 9-10 independent transformants. Northern blot analysis was as described in
Newman et a1. (1993). Hybridization probes corresponding to the Globin and GUS
coding regions were labeled using or-32P dCTP by the random-primed method of
F einberg and Vogelstein (1983). The hybridization probe corresponding to the E9
3’UTR was generated by transcribing the non-coding strand of this sequence in vitro
using T3 RNA polymerase in the presence of 0t-32P UTP, as described by Diehn et al.,
1998. Hybridization and washing conditions for the E9 RNA probe were also as
described in Diehn et al., 1998.

mRNA abundance was quantitated by measuring the intensity of radioactive

bands on Northern blots using a Molecular Dynamics Phosphorlmager.

AKNOWLEDGMENTS

I would like to thank Dr. Ambro van Hoof for identifying DST-like subdomains in the

SA UR-ACI 3’UTR.

118

REFERENCES

Bechtold,N., ElliS,J., and Pelletier,G. (1993). In planta Agrobacterium mediated gene
transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris 316,
1194-1199.

Diehn,S.H., Chiu,W.L., De Rocher,E.J., and Green,P.J. (1998). Premature
polyadenylation at multiple sites within a Bacillus thuringiensis toxin gene-coding
region. Plant Physiol. 117, 1433-1443.

F ang,R.-X., Nagy,F., Sivasubramaniam,S., and Chua,N.-H. (1989). Multiple cis
regulatory elements for maximal expression of the cauliflower mosaic virus 35S
promoter in transgenic plants. Plant Cell I, 141-150.

Feinberg,A.P. and Vogelstein,B. (1983). A technique for radiolabeling DNA restriction
endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.

Feldbrfigge,M., Sprenger,M., Dinkelbach,M., Yazaki,K., Harter,K., and Weisshaar,B.
(1994). Functional analysis of a 1i ght-responsive plant bZIP transcriptional regulator.
Plant Cell 6, 1607-1621.

Gil,P., Liu,Y., Orbovic,V., Verkamp,E., Poff,K.L., and Green,P.J. (1994).
Characterization of the auxin-inducible SA UR-A C1 gene for use as a molecular genetic
tool in Arabidopsis . Plant Physiol. 104, 777-784.

Gil,P. and Green,P.J. (1996). Multiple regions of the Arabidopsis SA UR-A CI gene
control trascript abundance: the 3' untranslated region functions as an mRNA instability
determinant. EMBO J. 15, 1678-1686.

Hunt,A.G. (1994). Messenger RNA 3' end formation in plants. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 45, 47-60.

Koncz,C. and Schell,J. (1986). The promoter of TL-DNA gene 5 controls the tissue-
specific expression of chimaeric genes carried by a novel type of A grobacterium binary
vector. Mol. Gen. Genet. 204, 383-396.

Kunkel,T.A., Roberts,J.D., and Zakour,R.A. (1987). Rapid and efficient Site-specific
mutagenesis without phenotypic selection. Methods Enzymol. 154, 367-382.

McClure,B.A., Hagen,G., Brown,C.S., Gee,M.A., and Guilfoyle,T.J. (1989).
Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.
Plant Cell 1, 229-239.

Newman,T.C., Ohme-Takagi,M., Taylor,C.B., and Green,P.J. (1993). DST sequences,
highly conserved among plant SA UR genes, target reporter transcripts for rapid decay in
tobacco. Plant Cell 5, 701-714.

119

Puissant,C. and Houdebine,L.-M. (1990). An improvement of the Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenol-chlorophorm extraction.
BioTechniques 8, 148-149.

Rogers,S.G., Klee,H.J., Horsch,R.B., and Fraley,R.T. (1987). Improved vectors for plant
transformation: expression cassette vectors and new selectable markers. Methods
Enzymol. 153, 253-277.

Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

Sullivan,M.L. and Green,P.J. (1996). Mutational analysis of the DST element in tobacco
cells and transgenic plants: Identification of residues critical for mRNA instability. RNA
2, 308-315.

Yamamoto,K.T., Mori,H., and Imaseki,H. (1992). cDNA cloning of indole—3-acetic acid-
regulated genes: Aux22 and SAUR from mung bean (Vigna radiata) hypocotyl tissue.
Plant Cell Physiol. 33, 93-97.

Ye,G.N., Stone,D., Pang,S.Z., Creely,W., Gonzalez,K., and Hinchee,M. (1999).
Arabidopsis ovule is the target for A grobacterium in planta vacuum infiltration
transformation. Plant J. 19, 249-257.

120

CHAPTER 2-1

USE OF POLY(G) INSERTIONS TO GAIN INSIGHT INTO THE STEPS INVOLVED

IN PLANT mRNA DEGRADATION

121

INTRODUCTION

Understanding the nature of the nucleolytic events that result in messenger RNA
(mRNA) degradation is a fundamental goal of mRNA turnover research. mRNAs have
two features that suggest the potential for multiple steps in the degradation process.
These features, the 7-methyl guanine cap and the poly(A) tail, are thought to protect the
5’ and 3’ ends of the message from degradation. One might suspect that Specific enzymes
or groups of enzymes have evolved that are responsible for removing these distinct
features. On the other hand, mRNA degradation may be a more stochastic process,
involving a series of endonucleases that digest RNA at non-Specific Sites. Unfortunately,
ascertaining the steps involved in the degradation of individual transcripts has not been
possible in most cases because once the process begins it is apparently completed very
rapidly. This supposition is verified by the common observation that only full-length
transcripts are detected on Northern blots. Therefore, the ability to analyze intermediates
in the mRNA decay process, which would provide a great deal of information about the
nucleolytic events involved in the process, has been limited.

There are a few cases of naturally occurring mRNA decay intermediates. For
example, intermediates in gro or and transferrin receptor mRNA degradation have been
analyzed in mammalian cells (Stoeckle, 1992; Binder et al., 1994). In the case of gro or,
an intermediate corresponding to the first 70% of the 5’ portion of the message
accumulates, but no 3’ intermediate is found. The 5’ intermediate could be caused by an
endoribonuclease or by a 3 ’ to 5’ exoribonuclease that stops at a particular Site. Two

degradation products are observed for the transferrin receptor, suggesting an

122

endonucleolytic cleavage event. Interestingly, the 3’ intermediate has been found to be
polyadenylated, indicating that the endonuclease activity precedes deadenylation.

In plant cells, there are two cases of naturally occurring mRNA decay
intermediates that have been thoroughly studied. The ribulose-1,5-bisphosphate
carboxylase small subunit RNA (SRS4) from soybean is degraded into a series of distinct
fragments (Thompson et al., 1992). The analysis of these fragments indicated that the
SRS4 transcript is degraded by a complex combination of endo- and exonucleolyitc
events. Degradation of the oat phytochrome A (PH YA) transcript also results in
fragments that were displayed on Northern blots. Analysis of these fragments led to the
proposal that while approximately 25% of the PHYA transcripts are deadenylated prior to
further degradation, most of the transcripts are degraded without prior deadenylation
(Higgs and Colbert, 1994). It is still unclear how the body of the PHYA transcript is
degraded and models involving endo- or exoribonucleases or combinations of both have
been proposed. These experiments have provided information about the degradation of
the SRS4 and PH YA transcripts, but information about the mechanisms responsible for
the turnover of other plant mRNAs is lacking.

A very fruitful alternative to analysis of naturally occurring mRNA decay
intermediates has been to trap intermediates by introducing stable secondary structures
into mRNAs. This approach was first used in Saccharomyces cerevisiae and has
contributed tremendously to the elucidation of a general pathway of mRNA degradation
in yeast (Vreken and Raue’, 1992; reviewed in Caponigro and Parker, 1996). The most
common insertion has been an 18 nucleotide poly-guanosine [poly(G)] tract, which is

capable of forming a stable secondary structure and is thought to block the progress of 5’

123

to 3’ or 3’ to 5’ exoribonucleases. By introducing poly(G) tracts into reporter genes, it
was found that in most cases, a deadenylated intermediate 5’ of the poly(G) tract was
stabilized. This provided strong evidence for a pathway that utilized a 5’ to 3’
exoribonuclease that degraded the body of the message after deadenylation. These
results were further strengthened by analyzing the accumulation of intermediates in yeast
mutants defective for exoribonucleases. For example, in strains with a deletion in the
XRNI gene, which encodes the predominant 5’ to 3’ exoribonuclease, this intermediate
does not accumulate. The major pathway of yeast mRNA degradation and the use of
poly(G) insertions to provide experimental evidence for this pathway are summarized in
Figure 2-1-1.

To get a first approximation of the kinds of mRNA degradation pathways that
might be involved in degrading plant messages, we designed reporter gene constructs that
contained poly(G) tracts and introduced them into plant cells from two plant Species.
Poly(G) tracts were inserted into reporter messages that might be degraded by distinct
mechanisms so that potentially diverse means of mRNA degradation could be analyzed.
The results of these experiments and their implications for the mechanisms of mRN A

degradation in plants will be described in this chapter.

124

 

m7Gpppl AUG - nAMAAso-roo
I deadenylation

m7GPPPLAUG 'nAo-io

I deadenylation-dependent
decapping

meg: Aue -nA...

I 5’ to 3’ degradation

 

 

 

o
6423,8119 mm
6“ B a“; exoribonuclease is

stalled

 

I intermediate accumulates

Figure 2-1-1. The effect of a poly(G) insertion on the major yeast mRNA degradation
pathway. “Pacmen” represent components of the yeast decay machinery as follows:
white, proposed deadenylating nuclease; light gray, decapping enzyme [Dcp1p, Dcp2p
(Beelman et al., 1996; Legrandeur and Parker, 1998; Dunckley and Parker, 1999)]; dark
gray, 5’ to 3’ exoribonuclease [Xmlp (Stevens, 1978; Muhlrad et al., 1994)]. The
intermediate that accumulates is trimmed to its 5’ end and contains an oligo(A) tail. In
strains with a deleted XRNI gene, this intermediate fails to accumulate. A version of this
figure appears in Caponigro and Parker (1996).

RESULTS

Poly(G) tracts were inserted into reporter constructs that had previously been
utilized to test mRNA sequences for their function as instability determinants in plant
cells (Newman et al., 1993; Ohme-Takagi et al., 1993; Gil and Green, 1996). Figure 2-1-
2 shows the basic structure of these reporters. These constructs provided the potential to

test whether rapid mRNA turnover mediated by a dimer of the DST element (DSTx2),

125

the AUUUA repeat, or the SAUR-AC1 3’ UTR would cause accumulation of different

poly(G) stabilized intermediates. These constructs (test genes) were introduced into
A grobacterium transfer DNAS (T-DNA) along with a B-glucuronidase (GUS) reference

gene.

nae-em

 

Test

 

 

 

 

% GUS I3Cfl Reference

Figure 2-1-2. Poly(G) tracts were inserted into a variety of plant reporter transcripts. The
cauliflower mosaic virus 35S promoter (358) was used to control the transcription of test
and reference genes. The human B-Globin (Globin) coding sequence was used as the test
reporter transcript in these studies. The reference gene, B-glucuronidase (GUS), was
included in the same binary vector with each of the test genes to facilitate normalization
of mRNA abundance data. The 3’UTRS of the pea ribulose 1,5-bisphosphate carboxylase
small subunit E9 (E9) and 3C (3C) genes were used as 3’UTRS for the test and reference
genes, respectively. G25 represents the 25 nucleotide poly(G) insertion; DST represents a
dimer of the DST element; AU represents the AUUUA repeat; SA UR represents the

SA UR-A C1 3 ’UTR. The right border (RB) of the T-DNA has been indicated by a black
rectangle. The T-DNA also includes the neomycin phosphotransferase cassette so that
kanamycin resistant transgenic plants or stably transformed tobacco cells could be selected.

These constructs were used to generate stably transformed tobacco cell lines and
transgenic Arabidopsis plants. RNA extracted from both systems was analyzed using

polyacrylarnide and agarose/fonnaldehyde gel blots. No detectable intermediates were

126

stabilized by the insertion of poly(G) tracts into any of these reporter transcripts in either
tobacco cells or Arabidopsis seedlings.

In all cases, only full-length Globin messages were detected. Hybridization
probes corresponding to the 5’ (Globin coding sequence) or the 3’ (E9 3’UTR) portions
of these reporters detected only full-length Globin transcripts. An example of one such
experiment is Shown in Figure 2-1-3. One possible explanation for the failure to detect
poly(G) intermediates is that our gel systems were not sufficient to detect the small
fragments that may have been stabilized. To address this concern, fragments of the
Globin reporter were generated in vitro by ribonuclease H (RNase H) digestion. RNase
H digests RNA at RNAzDNA duplexes such that one can produce specific RNA
fragments by incubating RNA with oligonucleotides complementary to sequences within
the transcript. This approach was used to generate fragments of the Globin reporter that
would correspond to poly(G) stabilized intermediates that accumulated 5’ or 3’ of the
insertion. In addition, a fragment was made in vitro that corresponded to an intermediate
that would have accumulated in vivo if the poly(G) tract blocked exonucleases that
attacked the transcript from the 5’ and 3’ ends simultaneously (Figure 2-1-3-A). As can
be seen in lanes 1, 2, and 3 of the Northern blot Shown in Figure 2-1-3-B, fragrrrents
generated in vitro corresponding to the possible poly(G) stabilized intermediates were
observed in the gel system. In contrast, only the full-length Globin transcript was
observed when the RNA sample was analyzed without prior RNase H treatment (lane 4).
This experiment indicated that although the gel system was adequate to detect poly(G)

stabilized intermediates, these intermediates did not accumulate in vivo.

127

A
4; «-

n"n eve—I

GUS . ,
Q < GlobinzG :E9

- O . < Globin:G::
poly(C) . < 62559

. ‘ G'25

 

Lane 1 2 3 4 5
RNaseH oligo AIB A B - -
POIY(G) + + + + -

Figure 2-1-3. Poly(G) stabilized intermediates do not accumulate in Arabidopsis. A. A
schematic diagram of the Globin reporter under analysis. Oligonucleotides for RNase H
digestion are indicated with arrows. B. Northern blot analysis. The top panel shows the
result of hybridization with radiolabeled GUS (reference gene) probe. The bottom panel
shows the result of hybridization with radiolabeled oligo(C) probe that detects all
transcripts containing the poly(G) tract. All lanes contain 20 pg of total RNA extracted
from pools of lO-day-old Arabidopsis seedlings. Lanes 1-3 of the bottom panel Show the
results of RNase H digestion of the Globin reporter. The oligonucleotide(s) used in each
experiment is indicated in the key below the panel. The predicted identity of each species
observed on the Northern blot is given on the right of the lower panel. RNA samples in
lanes 1-4 are fi'om plants expressing the transcript shown in A. The RNA sample in lane
5 is from plants expressing a similar reporter that lacks the poly(G) tract, indicating that
the hybridizing RNA species in lanes 1-4 were derived from the poly(G)-containing
transgene.

DISCUSSION

Insertion of poly(G) tracts into reporter transcripts was a potentially powerful and

rather obvious approach given the success of similar studies conducted in yeast (reviewed

128

in Caponigro and Parker, 1996). It is perhaps not surprising that similar constructs have
been introduced into a diverse array of organisms. However, successful stabilization of
poly(G) intermediates has only been reported thus far in yeast (for example, Vreken and
Raue, 1992) and Chlamydomonas reinhardtii (Gera and Baker, 1998; Drager et al., 1998
and 1999); poly(G) intermediates have not been detected in a multi-cellular organism. At
least three laboratories working on mammalian systems have attempted these
experiments with Similar results as those reported here for plants (L. Maquat, personal
communication; A.-B. Shyu, personal communication; G. Goodall, personal
communication). This lack of poly(G) intermediates may be indicative of differences in
mRNA decay mechanisms between simple and more complex eukaryotes.

There are several technical and biological explanations for the lack of poly(G)
intermediates in plant cells. For example, the poly(G) insertion may not form a strong
secondary structure when introduced into the cell. The use of an oligo(C) hybridization
probe in the experiment Shown in Figure 2-1-3, indicated that the poly(G) insertion was
present in the reporter transcripts, but that does not mean that the poly(G) insertion
formed a stable structure that could block exoribonucleases. It is possible that there are
RNA binding proteins in the plant cell that do not allow this structure to form.
Alternatively, the plant cell may have mRNA helicases that can unwind the poly(G)
structure allowing ribonucleases to proceed through it. An additional possibility is that
exoribonucleases homologous to the ones in yeast that cause poly(G) intermediates exist
in plant cells, but they are capable of degrading the poly(G) structure. Of course it is also
possible that such 5’ to 3’ exoribonucleases are not important for plant mRNA

degradation.

Recently, sequences Similar to the yeast XRNI gene have been deposited in the
Arabidopsis genome sequencing database. To begin to characterize the function of these
Arabidopsis XRN-like genes, cDNAs were cloned and introduced into yeast, all of them
led to the accumulation of poly(G) intermediates in yeast strains harboring an XRNI
deletion (James P. Kastenmayer and Pamela J. Green, unpublished). The role of these
genes in mRNA degradation in the plant cell has yet to be determined, however, these
results indicate that mRNaseS that can be stalled by poly(G) structures are present in
Arabidopsis.

The studies outlined in this chapter did not provide access to the steps involved in
degrading plant mRNAs. However, they point to a potential difference between mRNA
decay mechanisms between yeast and multi-cellular eukaryotes such as plants. The few
examples of mRNA decay intermediates that have been analyzed in plant and mammalian
cells also point to unique mechanisms for mRNA decay mechanisms in these systems. In
particular, the degradation of the gro or, transferrin receptor, SRS4, and PHYA transcripts
suggest a role for endonucleases in mRNA degradation. Endonucleases have not been
implicated in yeast mRNA degradation. One possibility is that poly(G) insertions fail to
block exoribonucleases in plants and animal cells because endoribonucleases, which may
be capable of degrading poly(G) tracts, are important for mRNA degradation in these
systems.

The presence of several sequences in the Arabidopsis sequence database that bear
similarity to components of the yeast mRNA decay machinery argues against vast
differences between yeast and plant mRNA decay mechanisms. In addition to the XRN-

like genes already mentioned, there are potential homologues of two members of the

130

decapping complex and of a human gene encoding a poly(A) ribonuclease.
Deadenylation and decapping are thought to be the first and rate-limiting steps,
respectively, of the major yeast mRNA decay pathway (Caponigro and Parker, 1996).
The role of these genes in mRNA degradation in plants has yet to be established,
however, the presence of these sequences in the Arabidopsis genome is intriguing and
points to the potential for some level of conservation among these pathways between
yeast and plants.

The constructs and transgenic plants generated for the work presented in this
chapter may be useful in analyzing the role of the mRNases. One of the goals of our
laboratory is to isolate mutants of Arabidopsis with T-DNA insertions in each of the
potential mRNaseS that have been identified through database searches. It is quite
possible that novel poly(G) intermediates will become apparent when one or more of

these genes are disrupted.

MATERIAL AND METHODS

Plasmid construction and plant transformation

Standard molecular cloning procedures were used to construct the chimeric genes
described in Figure 2-1-2. (Sambrook et al., 1989). The poly(G) tract was synthesized
using two oligonucleotides:

5 ’GATCTTCTAGAGGGGGGGGGGGGGGGGGGGGGGGGG-3 ’

5’GATCCCCCCCCCCCCCCCCCCCCCCCCCCTCTAGAA-3’. When annealed, these

131

oligonucleotides have a BamH I Site on the 5’ end and a BglII site on the 3’ end. This
facilitated the introduction of the poly(G) tract into the unique BamHI site present in pUC
derivatives with the structure: SacI-3SS-BglII-Globin-BamHI-E9-Cla1 or SacI-3SS-Bglll-
Globin-DSTx2-BamHl-E9 when an instability determinant was present. The BamHI Site
in SacI-3SS-BglII-Globin-poly(G)-BamHI-E9-Clal was filled in using T4 DNA
polymerase so that the SAUR-AC1 3’UTR could replace the E9 3’UTR following
digestion with ClaI and blunt-end ligation.

These test genes were transferred to a derivative of pMON505 which is a binary
vector that facilitates transformation of plant cells via Agrobacterium tumefaciens
(Rogers et al., 1987, Fang et al., 1989). The transfer DNA also contains a reference gene
with the structure 35S-GUS-3C. This test and reference strategy and the source of the
Globin and GUS coding regions were described by Newman et al., (1993).

Generation of and selection of transgenic Arabidopsis plants using the vacuum
infiltration method of A grobacterium-mediated transformation has been described (N.
Bechtold et al.,1993 and Web site: http://www.bch.msu.edu/pamgreen/vac.htm).

A grobacterium strain GV3101 C58Cl Rifr (pMP90) (Koncz and Schell, 1986) was
transformed with pMON505 derivatives by electroporation using the Gene-Pulser
(BioRad) according to the manufacturer’s guidelines.

Generation of and selection of stably transformed tobacco cells (Nicotiana
tabaccum cv Bright Yellow 2 [NT-1; An, 1985]) was as described previously by
Newman et a1. (1993). Agrobacterium strain LBA4404 was used in these experiments

and was transformed with pMON505 derivatives by electroporation.

132

RNA analysis

RNA was extracted from pools of transgenic tobacco calli, liquid tobacco
cultures, and pools of transgenic Arabidopsis seedlings using the method described by
Puissant and Houdebine (1990) with modifications described by Newman et al., (1993).

Northern blot analysis was as described in Newman et al., 1993 with the
following exceptions. Standard Northern blotting procedures were enhanced in order to
resolve potentially small poly(G) stabilized intermediates. 2.2% NuSieve 3:1 Agarose
(FMC, Inc.) / 2% formaldehyde gels were used to separate RNA. These gels were blotted
using standard methods. In some experiments polyacrylamide gels (6% acrylarnide, 7M
urea) were used to achieve enhanced resolution. These gels were run and transferred to
blots as described in Gera and Baker (1998).

RNA samples for the experiment shown in Figure 2-1-3 were extracted from
pools of lO-day—old Arabidopsis seedlings. These pools consisted of a total of ~1 00 T2
(self-fertilization progeny of primary transformants) seedlings from at least 10
independent transformants. T2 seedlings were grown on seed selection media which
consisted of 4.3 g/L Murashige and Skoog salt mixture (GibcoBRL), BS vitamin mixture
(100 mg/l myo-inositol, 10 mg/l thiamine hydrochloride, 1 mg/l nicotinic acid, 1 mg/l
pyriodoxine), 1% sucrose, 0.5 g/L MES at pH 5.7, 0.8% phytagar, and 50 pg/ml

kanamycin. Plates were kept in incubators set at 16 hours light (125 uE/mz) /8 hours

dark, 21°C.

133

RNase H treatment

Oligonucleotides complementary to the 3 ’ end of the Globin coding sequence (pg-
244) or the 5’ end of the E9 3’ UTR (pg-316) were used to digest the reporter transcript at
specific Sites in vitro as described in Diehn et al. (1998). Briefly, 20 pg of total RNA was
incubated with 2 ug of each oligonucleotide at 65°C for 30 minutes in a 400 ml water
bath. To anneal the oligonucleotides to their cognate mRNA, the water bath was allowed
to cool to 30°C. The reactions were then placed at room temperature for 5 minutes.
RNase H digestions were done at 37°C in a total volume of 50 “Is that included 2 units of
RNase H and RNase H reaction buffer (4 mM Tris-HCI, pH 8.0, 10 mM MgCl2, 20 mM

KC], 1 mM DTT) (GibcoBRL).

ACKNOWLEDGEMENTS

I started this project during my rotation in the Green Laboratory under the careful
tutelage of Dr. Michael Sullivan. I’d like to thank Mike for his incredible patience and

for all he taught me during that ten-week period.

134

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Bechtold,N., ElliS,J., and Pelletier,G. (1993). In planta Agrobacterium mediated gene
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Beelman,C.A., Stevens,A., Caponigro,G., LaGrandeur,T.E., Hatfield,L., Fortner,D.M.,
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Binder,R., Horowitz,J.A., Basilion,J.P., Koeller,D.M., Klausner,R.D., and Harford,J.B.
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Caponigro,G. and Parker,R. (1996). Mechanisms and control of mRNA turnover in
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Diehn,S.H., Chiu,W.L., De Rocher,E.J., and Green,P.J. (1998). Premature
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Drager,R.G., Girard-Bascou,J., Choquet,Y., Kindle,K.L., and Stern,D.B. (1998). In vivo
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Gera,J.F. and Baker,E.J. (1998). Deadenylation-dependent and -independent decay
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control trascript abundance: the 3' untranslated region functions as an mRNA instability
determinant. EMBO J. 15, 1678-1686.

Higgs,D.C. and Colbert,J.T. (1994). Oat phytochrome A mRNA degradation appears to
occur via two distinct pathways. Plant Cell 6, 1007-1019.

Koncz,C. and Schell,J. (1986). The promoter of TL-DNA gene 5 controls the tissue-
specific expression of chimaeric genes carried by a novel type of Agrobacterium binary
vector. Mol. Gen. Genet. 204, 383-396.

LaGrandeur,T.E. and Parker,R. (1998). Isolation and characterization of Dcplp, the yeast
mRNA decapping enzyme. EMBO J. 17, 1487-1496.

Muhlrad,D., Decker,C.J., and Parker,R. (1994). Deadenylation of the unstable mRNA
encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the
transcript. Genes Dev. 8, 855-866.

Newman,T.C., Ohme-Takagi,M., Taylor,C.B., and Green,P.J. (1993). DST sequences,
highly conserved among plant SA UR genes, target reporter transcripts for rapid decay in
tobacco. Plant Cell 5, 701-714.

Ohme—Takagi,M., Taylor,C.B., Newman,T.C., and Green,P.J. (1993). The effect of
sequences with high AU content on mRNA stability in tobacco. Proc. Natl. Acad. Sci.
USA 90,11811-11815.

Puissant,C. and Houdebine,L.-M. (1990). An improvement of the Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenoI-chlorophorm extraction.
BioTechniques 8, 148-149.

Rogers,S.G., Klee,H.J., Horsch,R.B., and Fraley,R.T. (1987). Improved vectors for plant
transformation: expression cassette vectors and new selectable markers. Methods
Enzymol. 153, 253-277.

Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

Stevens,A. (1978). An exoribonuclease from Saccharomyces cerevisiae: effect of
modifications of 5' end groups on the hydrolysis of substrates to 5' mononucleotides.
Biochem. Biophys. Res Commun. 81, 656-661.

Stoeckle,M.Y. (1992). Removal of a 3' non-coding sequence is an initial step in
degradation of groa mRNA and is regulated by interleukin- 1. Nucleic Acids Res. 20,
1123-1127.

Thompson,D.M., Tanzer,M.M., and Meagher,R.B. (1992). Degradation products of the
mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean
and transgenic petunia. Plant Cell 4, 47-58.

136

Vreken,P. and Raue’,H.A. (1992). The rate—limiting step in yeast PGKI mRNA
degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.
Mol. Cell. Biol. 12, 2986-2996.

137

CHAPTER 2-2

CLONING AND INITIAL CHARACTERIZATION OF A

POLY(A) RIBONUCLEASE FROM ARABIDOPSIS

138

INTRODUCTION

With few exceptions, the poly(A) tail is common to all eukaryotic messenger
RNA (mRNA) molecules. There is also recent evidence that this structure is present on
many bacterial RNAS (reviewed in Carpousis et al., 1999). The poly(A) tail appears to be
involved in controlling mRNA stability in prokaryotes and eukaryotes; in prokaryotes it
is thought to be a signal for rapid mRNA degradation while in eukaryotes it protects
mRNA from degradation (reviewed in Jacobson, 1996). The poly(A) tail is also an
enhancer of translation in eukaryotes (reviewed in Gallie, 1998). The role of the poly(A)
tail in controlling mRNA function has made it a target for developmental regulation. For
example, in Xenopus, poly(A) tails are added, or removed to promote or inhibit
expression of certain genes during oocyte maturation (Fox and Wickens, 1990; Vamum
et al., 1992; Sheets et al., 1995). The poly(A) tail is also a point of post-transcriptional
control of gene expression in general, and in yeast it has been found that deadenylation
triggers the degradation of the majority of cytoplasmic messages (reviewed in Caponigro
and Parker, 1996).

The major yeast mRNA degradation pathway is thought to begin with
deadenylation which is followed by cleavage of the of the 7-methyl guanosine cap
(decapping) and eventual degradation of the body of the mRNA by a 5’ to 3 ’
exoribonuclease. These three steps are dependent upon one another such that decapping
cannot occur without deadenylation and 5’ to 3’ degradation will not occur without prior
decapping (reviewed in Caponigro and Parker, 1996). Nucleases that are responsible for

decapping and degradation of the body of the transcript have been cloned and their role in

139

the major yeast mRNA degradation pathway has been demonstrated genetically and
biochemically (Muhlrad et al., 1994; Beelman et al., 1996; Legrandeur and Parker, 1998;
Dunckley and Parker, 1999). In contrast, the enzyme(s) responsible for complete
degradation of the poly(A) tail has not been identified. Two candidates for this activity
have been studied in yeast. First, a poly(A) binding-protein (PAB)-dependent poly(A)
nuclease was cloned, however, recently it was Shown that this activity is most likely
responsible for trimming poly(A) tails to their mature length (Brown and Sachs, 1998).
The exosome, an additional candidate, is a recently described complex of ~10 3’ to 5’
exoribonucleases involved in degradation of yeast mRNA, but the substrate for this
complex is thought to be deadenylated mRNA (reviewed in van Hoof and Parker, 1999).
Therefore the deadenylating nuclease remains unknown in yeast.

A 3’ to 5’ exoribonuclease that preferentially degrades poly(A) was purified from
calf thymus (Komer and Wahle, 1997). This enzyme, was Shown to have characteristics
that identify is a candidate poly(A) riboguclease and was called PARN. In vitro, PARN
is Mg2+-dependent, and at low salt concentrations was dependent on Spermidine or PAB.
At physiological salt concentrations, sperrnidine activation was not as strong and PAB
was inhibitory (Komer and Wahle, 1997). Incubation of PARN plus PAB with
radiolabeled capped and polyadenylated mRNA Species results in phased degradation
products that are consistent with protection of the poly(A) tail by PAB (Komer and
Wahle, 1997). A cDNA clone corresponding the human homologue was subsequently
obtained from the human genome sequencing project and is referred to as HuPARN
(Komer et al., 1998). The protein encoded by the HuPARN cDNA was expressed in

Escherichia coli and purified and was found to have similar properties to the enzyme

140

originally purified from calf thymus. Experiments conducted in Xenopus oocytes
implicate HuPARN as a potential deadenylating nuclease in vivo (Komer et al., 1998).
A sequence similar to HuPARN was identified in the Arabidopsis genomic
sequence by Komer et al. (1998). To determine if AtPARN (for Arabidopsis thaliana
PARN-like gene) is involved in mRNA degradation in plants, a cDNA corresponding to
this gene was cloned and we have begun to characterize its function. These studies have
the potential to answer many interesting questions about PARN in particular and mRNA
degradation in general. For example, it is not known what role if any this type of protein
may play in general mRNA degradation mechanisms. Is PARN responsible for
triggering general mRNA degradation by removal of poly(A) tails? Is AtPARN
important for post-transcriptional control of gene expression in plants? Does
conservation of PARN indicate deadenylation plays an important role in plant mRNA
decay pathways as it does in yeast? By applying the molecular genetic techniques
available in Arabidopsis to the study of AtPARN, we may answer some of these important

questions.

RESULTS

The RNaseD family and cloning of AtPARN

Sequence analysis revealed that HuPARN was a member of an ancient gene
family of RNases named after it’s founding member, RNaseD (Komer et al., 1998; Mian,
1997; Moser et al., 1997). RNaseD is involved in processing of tRNAs in E. coli

(reviewed in Deutscher, 1993). This family includes PAN2 from yeast which is a subunit

I41

of the PAB-dependent poly(A) trimming activity described above. The feature that unites
this gene family are three 3’ to 5’ exoribonuclease (Exo) domains which contain acidic
residues involved in the coordination of metal ions necessary for nuclease activity
(Bamad et al., 1989; Joyce and Steitz, 1994). This catalytic domain iS also common to
the proofreading domain of E. coli DNA polymerase I. Mian used a hidden Markof
model to find RNaseD family members and to characterize important amino acids in
these proteins (1997). This algorithm allows one to find specific domains within
unrelated polypeptides; outside of the Exo domains many of the RNaseD family members
are not similar. For example, there are several open reading frames within the yeast
genome that lack Exo domains, but are more similar to HuPARN than is PAN2, an
RNaseD family member (Komer et al., 1998).

A region of the Arabidopsis genome was identified that bore Significant similarity
to the HuPARN sequence. Among the conserved residues were the three Exo domains,
the hallmark of the RNaseD family. The genomic sequence was used to design primers
for rapid amplification of cDNA ends (RACE) by polymerase chain reaction (PCR). The
predicted amino acid sequence of the resulting clone is Shown in an alignment with the
HuPARN amino acid sequence in Figure 2-2-1. AtPARN and HuPARN are 21%
identical at the amino acid level. Interestingly, AtPARN has a 38 amino acid N-terrninal
extension. Eleven out of 38 residues in this N-terminal sequence are serine or threonine,
amino acids that are often over-represented in N-terrninal chloroplast targeting signals.
However, the AtPARN polypeptide failed to enter chloroplasts in a targeting assay (John

Froehlich, data not Shown).

142

WPLR LVCSP AAETVTTSTAASAEAAFP TTLNDLRSL
:0 O ::::...:O O O O

 

 

 

FVSGRHNFFVFPRQELTFDPPAHEFLCQTTSMDFLAKYQFDFNTCIHEGISYLBRREEEB
... ::.:::. .. . : .:.::..:.::::. 33:: ...:: :z...::

YITKSFNFYVFPKP - FNRSSPDVKFVCQSSSIDFLASQGFDFNKVPRNGI PMQII- -

ASKRLKMLHGEDGIDSSGETEBLKLVRLADVLPAARMEKLLNIWRSGLLHGGNLSSIFPR
00:. O : ...: :0 : CO. C : C. O. O. .

--RQLREQYDEKRSQANGAGA-LSYVSPN----TSXCPVTIPBDQKKPI ----- DQVVER

ISNGSNOSMETVFHHMRPALSLKGFTSHQLRVLNSVLRKHPGDLVYIHSNDKSSSSRDIV
: O 3: :0 30:. :0 : ... 0.: O. .0... C C. 3 ::
IED-LLQSEEN ------ KNLDLEPCTGFQRKLIYQTLSWKYPKGIHVITLITEKKIRYIV

 

  

I---SKVDEEE--RKRREQQKHAKBQEELNDKVGPSRVIHAIANS

Exmll
vrtsmcpnps'rmrvnsrumpxrmxnmomssrsnssussmp
O C: III. .8 0 :g ..z::.. 0.0:. O .0: :0 O 8
TVHQFYCPLPADLSIFREMTTCVPPRLLDGKLMASTQPF---KDIINNTSLA8LIKRL-R

 

QIE?SSRSSDSPLQQRVNIDVEIDNVRC‘ ”L;' ;_"w 6 IPAQACNHLGPD
. :0 O O: C O I. .0. “I I

memems -AEGI'PSYD‘1‘ASEQL --------- " ' - 1’ encrrsnmm-s

Exolll
PKQHSQLDDPAQNIKLEKYINRLYLSWT -------- RGDIIDLRTGH----SN1DNWRV8

PLSPPKIHVSARSKLIEPFPNKLIEMRVNDIPYLNLEGPDLQPKRDHVLHVTFPXIWKI8

  

Kr ----- KYENIVLIW-NFPRKLKARGIKBCICKAFGSASVTSVYHVDDSAVPVLFKNS!
3: o : o o o o I o :0... o :00. 3 o. :0 I

DLYQLPSAPGNIQISW1DDTSAFVSLSQPEQVKIAVNTSKYAESYRIQTYARYMGRKQIB

KQIKRKWTEDSWKEADSKRLNPQCIPYTLQNHYYRNNSFTAPSTVGKRNLSPSQEEAGLE

DQAITVGVKSRTRPNAQCETBTRE--ENTVTVTHKASDLIDAFLANR----VEVETATSN

DGVSGEISDTELEOTDSCAEPLSEGRKKAKKLKRMKKELSPIGSISKNSPATLPEVPDTW

Figure 2-2-1. Sequence alignment of the AtPARN and HuPARN amino acid sequences.
The AtPARN polypeptide, listed as the first line, is predicted to have 689 amino acids;
HuPARN consists of 639 amino acids. The N-terminal extension found in AtPARN is
boxed. Three Exo domains are also boxed and key residues are shaded. Sequence
identity is indicated by a :, chemically similar residues (.) are also indicated.

143

RnaseD family members have diverse functions (Deutscher, 1993, Moser et al.,
1997). Therefore it may be important to analyze the relative sequence similarity between
different family members in order to make functional approximations. AtPARN and
HuPARN share Similarity outside of the Exo domains and are more similar to each other
than they are two other members of the RNaseD family. BLAST (basic local alignment
search tool) searches using the predicted AtPARN amino acid sequence find only
HuPARN and a gene from Caenorhabditis elegans (K10C81, also described in Komer et
al., 1998). AtPARN is not Significantly Similar with other members of the RNaseD
family, including PAN2, in these searches. The close relationship between HuPARN and

AtPARN relative to other members of the RNaseD family may indicate functional

similarity between these proteins.

AtPARN is expressed throughout the plant

To determine the expression pattern of A tPARN in Arabidopsis plants, Northern
blot analysis was performed on total RNA isolated from roots, stems, leaves, and flowers.

AS Figure 2-2-2 indicates, the AtPARN mRNA was found in all of these plant organs.

or {'3

a g g 9

8 a 8 3

C U) ...I ll.

" Q ‘ AtPARN Figure 2-2-2. AtPARN mRNA is found throughout
the plant. A Northern blot containing 20 pg of total
RNA from roots, leaves, stems, and flowers of

4,; . w 4 elF4-A Arabidopsis plants was hybridized sequentially with

AtPARN and eIF4-A probes. A photograph of the
ethidium-bromide stained gel is included to
highlight differences in abundance of chloroplast

4 rRNA ,
,2; RNA in these samples.

 

144

The Size of the AtPARN transcript was approximately 2 Kb, about that of the
cDNA clone which was 2193 base-pairs excluding the poly(A) tail. Northern blot
analysis indicated that AtPARN mRNA was more abundant in roots and flowers than in
leaves and stems (Figure 2-2-2). These data must be interpreted with caution, however,
because the differences in AtPARN mRNA abundance may reflect differences in loading
due to the smaller amount of chloroplast rRNA in roots and flowers. The eIF4-A
transcript abundance was to be used as loading control, however, the abundance of this

message also varied in these samples.

Characterization of AtPARN activity in vitro

To analyze the enzymatic activity of the AtPARN polypeptide, the coding
sequence was expressed in E. coli as an N-terminal six-histidine fusion. This construct
facilitated the partial purification of the AtPARN polypeptide using a Ni2+-NTA column.
Denaturing polyacrylamide gel electrophoresis showed that two major proteins were
eluted from the Ni2+-NTA column, only one of which cross-reacted with anti—
pentahistidine antibodies (data not Shown). These proteins were approximately 70 kd,
matching the predicted size of AtPARN, the smaller one may be the result of an N-
terminal degradation that occurred after purification (Figure 2-2-3-A).

Poly(A) ribonuclease activity was analyzed using a trichloroacetic acid (TCA)
precipitation assay that monitors the release of mononucleotides from a uniformly
radiolabeled poly(A) substrate following incubation with AtPARN. Preliminary assays
indicate that AtPARN has poly(A) ribonuclease activity (Figure 2-2-3-B). Thus far, only

conditions optimal for HuPARN have been used in these assays.

145

*3
3 B
|.I.l

 

 

E coll
Substrate
only
HuPARN
AtPARN

Figure 2-2-3. AtPARN has poly(A) ribonuclease activity. A. A denaturing
polyacrylamide gel was loaded with approximately equal amounts of protein from the
flow through (FT), the first wash (W1), the second wash (W2), and the eluate of a Ni2+-
NTA column. A photograph of the Coomassie stained gel is shown. B. The AtPARN
eluate was tested using the TCA PARN assay (described in materials and methods). The
average counts per minute (CPM) detected in two experiments is plotted i the standard
deviation. The eluate from BL21(DE3)pLysS cells (E. coli) that were not expressing
AtPARN and the assay mixture with no protein added (Substrate only) served as negative
controls. HuPARN was used as a positive control.

DISCUSSION

Deadenylation is the first step in the degradation of many yeast and mammalian
mRNAs and the rate of deadenylation is important in determining mRNA stability in
these systems (Shyu et al., 1990; Muhlrad et al., 1994, Ross, 1995; Caponigro and Parker,
1996). There is evidence that deadenylation may be involved in the degradation of at
least one plant message, the oat phytochrome A transcript (Higgs and Colbert, 1994). A

thorough study of poly(A) degrading activities in plants is necessary to determine the role

146

of this process in plant mRNA stability. In addition, studying a poly(A) ribonuclease in
plants may provide important information about the mechanism of deadenylation in other
eukaryotes.

Eukaryotic mRNA turnover pathways have been studied almost exclusively in
yeast. In Saccharomyces cerevisiae, genes have been identified that encode every enzyme
in the major mRNA decay pathway except the deadenylating nuclease. Thus far, the best
candidate in any eukaryote for such an activity is HuPARN, a 3’ to 5’ exoribonuclease
that degrades poly(A) preferentially (Komer and Wahle, 1997, Komer et al., 1998). The
characteristics of this protein in vitro are consistent with a role in deadenylation
(discussed above, Komer et al., 1997). Perhaps the best evidence that HuPARN may
play a role in deadenylation of mRNA comes from studies in Xenopus oocytes. A
deadenylating nuclease activity that catalyzes the default removal of poly(A) tails in
mature oocytes was identified almost ten years ago (Varnum et al., 1990). Purification of
a protein responsible for this activity and cloning of the gene that encodes it revealed
strong Similarity to HuPARN (Komer et al., 1998). Antibodies directed against HuPARN
blocked the default deadenylation of transcripts in mature oocytes and ectopic expression
of HuPARN promoted deadenylation in enucleated oocytes lacking Xenopus PARN.
These experiments indicated that HuPARN is a deadenylase in vivo (Komer et al., 1998).

We have cloned a cDNA from Arabidopsis that bears significant Similarity to
HuPARN. Initial experiments indicate that this gene, called AtPARN, encodes a protein
with poly(A) nuclease activity in vitro. Thus far only assay conditions that were optimal

for HuPARN activity have been tested. The AtPARN mRNA is expressed throughout the

147

plant as might be expected for a gene involved in a ubiquitous process such as
deadenylation of mRNA.

Future experiments will address the role of this gene in mRN A degradation in
Arabidopsis. We have initiated the search for a transfer-DNA (T-DNA) insertion in
AtPARN. A collection of ~68,000 plants that collectively have ~100,000 T-DNA inserts
Spread throughout the genome has been screened by PCR using primers corresponding to
the borders of the T-DNA and the AtPARN genomic sequence (Hirsch et al., 1998).
Initially, 30 large pools containing DNA from the entire collection were screened to
identify those with inserts in AtPARN. These pools must be dcconvoluted through a
series of sub-pools in order to find single plants with the T-DNAS of interest. The PCR
products corresponding to five T-DNA inserts in AtPARN have been sequenced. One T-
DNA is inserted just after the first Exo domain within the third cxon of the AtPARN
genomic sequence. A pool of nine plants has been identified that contains plants with
this T-DNA insertion. The T-DNA is ~8 Kb long, so insertions will likely result in a null
mutation. Based on database searches and Southern blot analysis (data not Shown),

A tPARN appears to be the only PARN-like sequence in the Arabidopsis genome.
Therefore, a mutant in this gene Should be informative. Of course if AtPARN is
important for the control of mRNA stability in plants, loss of function may be lethal. One
of the advantages of isolating mutants by PCR screening is that heterozygotes can be
identified if the mutation is recessive. Analysis of the progeny of these heterozygous
plants allows one to determine if a mutation is lethal, an opportunity not available to
classical geneticists. The chief goal of these studies will be to study the effect on poly(A)

tail length in these mutants and analyze any phenotypic changes that become apparent.

148

A fusion of HuPARN to the green fluorescent protein (GFP) was localized to the
nucleus and cytoplasm of Cos-1 cells (Komer et al., 1998). It is unclear why HuPARN
would be found in the nucleus in COS-1 cells, but one would expect a deadenylase
involved in mRNA degradation to be found in the cytoplasm. In addition, Xenopus
PARN isoforms are found in the nucleus and in the cytoplasm of mature oocytes (Komer
et al., 1998). In Xenopus, PARN activity is thought to be regulated by sequestration in
the nucleus. One interesting question raised by these studies is whether PARN is a
specialized activity involved in the default deadenylation of mRNAs developing zygotes
or whether it is involved in general mRNA degradation. The initial purification of
mammalian PARN from calf thymus and the expression of HuPARN mRNA in many
different types of human cells would suggest that this is not true of HuPARN, and
whether this is true of Xenopus PARN remains to be tested (Komer et al., 1998).
AtPARN is expressed throughout the plant, but we do not yet know where it is localized
within the plant cell or whether it is expressed preferentially in certain cell types. We
have initiated these localization studies by introducing an N-terrninal GFP fusion into
Arabidopsis plants.

Genetic strategies are limited in mammalian cells and in Xenopus, therefore, the
ability to test the function of AtPARN directly by isolating a T-DNA insertion mutant
may provide us with the unique opportunity to address the role of this type of enzyme in

cytoplasmic mRNA degradation in somatic cells.

149

MATERIALS AND METHODS

Cloning of AtPARN

Standard cloning procedures were used as described in Sambrook et al. (1989).
Two products of RACE experiments corresponding to the 5’ and 3’ ends of the AtPARN
cDNA were generated using primers complementary to the AtPARN genomic sequence
and to the adapter sequence that was 1i gated to the ends of cDNAs from Arabidopsis
seedling mRNA. The details of this protocol are described in the package insert of the
Marathon cDNA kit (Clonetcch). The 5’ product consisted of the first 1.5 kb of the
AtPARN cDNA and was amplified using primer pg-444 (5’-
TGAAGTCAAAACCGAGATGATTGC -3’). This product was inserted into the EcoRV
site of a derivative of pBlueskript SKII(-) called p948, the resulting clone was called
p1843. The 3’ product consisted of the latter 0.9 kb of the AtPARN cDNA and was
amplified using primer pg-609 (5’-AGGTTGCATCTTTGCGCAGGC -3’). This product
was inserted into the EcoRV site of p948, the resulting clone was called p1920. The
overlap Shared by these two PCR products contained a unique Sphl Site that allowed the
two products to be joined, creating p1932.

The final cDNA clone was sequenced and checked against two available genomic
sequences. No errors in the cDNA were found. The AtPARN Genomic region consists
of ~3000 Kb on bacterial artificial chromosome f14j16 which has been mapped to
chromosome 1 near position 80. Comparison of the cDNA with the genomic sequence

revealed that AtPARN consists of 7 cxons.

150

 

Analysis of A {PARN expression

Total RNA was isolated from the leaves, stems, and flowers of mature (6 week
old) Arabidopsis (Col-0) plants. Root RNA was prepared from the roots of seedlings
grown in liquid culture for 3 weeks (2.3% Gamborg’s medium [GibcoBRL, Inc.], pH
5.0). RNA was prepared using the method described by Puissant and Houdebine (1990)
with modifications described by Newman et al., (1993). Northern blot analysis was as
described in Newman et al. (1993). The use of the translation initiation factor, eIF4-A, as
a loading control for Northern blots was as described by Taylor et al., (1993).
Hybridization probes corresponding to the AtPARN and eIF4-A coding regions were
labeled using 0t-32P dCTP by the random-primed method of Feinberg and Vogelstein

(1983).

Expression of AtPARN in E. coli

The vector used to express HuPARN in E. coli (pGMMCS) was obtained from
Wahle and coworkers (Komer et al., 1998). This is a derivative of the pET vectors
(Novagen). To fuse Six histidine residues to the N-terrninus of the AtPARN coding
sequence and to facilitate cloning into pGMMCS, the primers pg-718 (5’- T GCC ATG
GCT CAC CAT CAC CAT CAC CATGTC GAC A_TG CGC CGG CAC AAG CGA TG —

3’) and pg-727 (5’ — CG TGC TCG AGT TAA TTA CTC GTA GCA GIT TCG - 3’)

151

were used to amplify the AtPARN coding sequence by PCR. Stop and start codons are
underlined, the codons encoding the six histidine residues are italicized. The resulting
PCR product was inserted at the EcoRV site of p948 creating pl938. The AtPARN
coding region was sequenced and no errors were found. An Ncol, Xbal fragment of this
clone was then introduced into pGMMCS creating p1949, the AtPARN E. coli expression
vector.

BL21(DE3)pLysS (Novagen) cells were transformed with p1949 by
electroporation using the Gene-Pulser (BioRad) according to the manufacturer’s
guidelines. Induction of AtPARN expression was carried out in LB medium
supplemented with chloramphenicol (33 pg/ml), ampicillin (100 pg/ml) and IPTG (1
mM) as described on pages 46 and 47 of the QIAexprcssionist (1997, Qiagen Inc.). 10
ml cultures from three clones that expressed AtPARN to high levels were combined and a
soluble lyste was prepared as described on page 63 of the QIAexprcssionist (1997,
Qiagen Inc.). AtPARN was purified using a Ni2+-NTA (nitrlotriacctic acid) resin that was
incubated with p1949 soluble lysates in a batch culture at 4°C as described on pages 66
and 67 of the QIAexprcssionist (1997, Qiagen incorporated). The denaturing
polyacrylamide gel shown in Figure 2-2-3-A consisted of 10% polyacrylamide and 5%
SDS. Lysates from BL21(DE3)pLysS (Novagen) cultures were prepared in parallel and
were resolved using NiZ+-NTA resin so that eluates from these columns could be used as

a negative control in PARN assays.

152

TCA PARN assay

This assay monitors the release of TCA-soluble products from homogeneously
labeled poly(A). The substrate was prepared as described in Brown et al.(l996). The
reaction mixture consisted of 500 units of yeast poly(A) polymerase (U .S. Biochemicals),
50 pCi (1:32P rATP, 0.5 pM l2 nucleotide oligo(A) (obtained from Dharmacon Research,
Inc.), 167 mM rATP, 1X poly(A) polymerase buffer (U.S. Biochemical). The reaction
was incubated at 30°C for 60 minutes. Polyacrylamide gel electrophoresis showed that
labeled poly(A) was greater than 1 Kb in length. Unincorporated nucleotides were
removed using a spin column (S-200, Phannacia).

100,000 CPM of the poly(A) substrate was incubated with 10 pls of BL21 eluate,
10 pls of AtPARN eluate, or 1 pl of HuPARN or with the PARN assay mixture alone.
The concentrations of AtPARN and HuPARN were not determined. The assay mixture
and conditions were as described in Komer and Wahle (1997) and modified based on
Brown et al. (1996). Briefly, eluates were added to a reaction mixture consisting of 2mM
sperrnidine plus dilution buffer (5mM Hepes, pH 7.4; 2mM MgC12; 14.4mM B-
mercaptoethanol) in a total volume of 50 pls and were incubated at 37°C for 30 minutes.
200 pls of cold 20% TCA was then added and polynucleotides were precipitated at -20°C
for 10 minutes. Pellets were spun down and 100 pls of the supernatant was counted by
liquid scintillation. Two experiments that were conducted one after the other are reported

in Figure 2-2-3-B.

153

 

ACKNOWLEDGEMENTS

I would like to thank Drs. Ambro van Hoof and Roy Parker of The University of
Arizona who made us aware of the AtPARN genomic sequence before publication of
Komer ct a1. (1998); and Dr. Eva Devlin in Dr. Elmar Wahlc’s group at the Universitiit
Hallc Institut fiir Biochemie, who provided pGMMCS and an aliquot of HuPARN. I
thank Jonathan Vo gel for his great interest in this project and for conducting the TCA
PARN assays reported in Figure 2-2-3-B. I would also like to thank Prectrnoninder
Lidder who constructed a GFP-AtPARN fusion protein and iS currently analyzing its
localization in transgenic Arabidopsis. Thanks also to James Kastenmayer who
generated the cDNA library that was used to clone AtPARN and Miguel A. Pérez-Amador

who generated the Northern blot used for Figure 2-2-2.

154

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