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Nuclear galectins and their role in pre-mRNA splicing

presented by

Weizhong Wang

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6/07 p ICIRC/DateOueindd—pt

Nuclear galectins and their role in pre-mRNA
splicing

By

Weizhong Wang

A DISSERTATION

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2006

Abstract

Nuclear galectins and their role in pre—mRNA splicing

By

Weizhong Wang

Galectins are a family of carbohydrate binding proteins that share conserved
sequence elements in their carbohydrate-binding sites. In previous studies, we have
documented that galectin-1 and galectin-3 are necessary and redundant splicing factors in
an in vitro splicing system.

We wanted to know whether the splicing activity of galectins depends on the
carbohydrate binding activity. To address this question, we obtained mutant galectin-l
construct from Hirabayashi’s lab. Residue Asn46 is critical for carbohydrate binding
activity of galectin-1. By mutating this residue to Asp, the carbohydrate binding activity
of galectin-1 was abolished. This mutant was tested along with the wild type galectin-1 in
depletion and reconstitution experiments. The results show that the mutant galectin
reconstituted splicing as well as the wild type. This allows us to draw the conclusion that
the carbohydrate binding activity of galectin-1 is separate from its splicing activity.

The next goal was to determine how galectins are involved in the splicing process.
Our hypothesis was that galectins associate with the spliceosome through protein-protein
interactions. Immunoprecipitation was used to test this hypothesis. Pre-mRNA was added
to HeLa nuclear extracts to assemble spliceosomes, then they were mixed with antibody
against galectin-1 or galectin-3 and the bound fractions were isolated and subjected to

RNA or protein analysis. The results show that both antibodies can immunoprecipitate

spliceosomal RNAs and spliceosomal protein components. We conclude that galectins
physically associate with spliceosomes throughout the splicing process. To further
characterize this association, splicing reactions were sequentially immunoprecipitated
with galectin antibodies. We found that galectin-1 containing spliceosomes did not
contain galectin-3 and vice versa. These data provided an explanation for the functional
redundancy of galectin-1 and galectin-3 in splicing activity. Another finding is that the
association of galectins with the spliceosomes can be disrupted easily by ionic conditions.
Since most of the proteomic studies on spliceosomes used stringent ionic conditions, this
result could explain why galectins haven’t been found in these studies. We conclude that
galectin-1 and -3 are directly associated with splicing complexes throughout the splicing
pathway in a mutually exclusive manner and they bind a common splicing partner
through weak protein-protein interactions.

Since galectins associate with spliceosomes, it is intriguing to analyze whether
they also associate with endogenous nuclear complexes. Using similar
immnoprecipitation techniques, we confirmed our hypothesis. Galectin—l and -3 are both
components of nuclear snRNP containing complexes. This association does share some
common features as the galectins’ association with spliceosome. They are both sensitive
to ionic strength. They only incorporate one type of galectin into the complex and
mutually exclude the other. Pre-formed galectin containing complex could not be
disturbed by carbohydrate ligands. In the future, galectin containing nuclear complexes

might be the tools to reveal the mechanism of galectin incorporating into spliceosomes.

Acknowledgments

First and foremost, I would like to thank my mentor, Dr. Ronald Patterson. His
support, instruction, and encouragement are of great value for me both academically and
personally. Without him, I could hardly finish my PhD research. I will forever remember
him as a kind and intellectual adviser.

Next, I’d like to thank the members of my graduate study committee, Dr. John
Wang, Dr. Richard Schwartz and Dr. Jerry Dodgson. They gave me precious advice,
helped to solve problems encountered and guided me through my research study.

I also want to thank all my friends in the department and collaborating lab,
including Jung Park, Kevin Haudek, Patty Voss, Richard Gray, Sara Santos, Shibani
Mukherjee, Xiaoyu Wang, Lianjie Li and many others. Their friendship and support will
be with me forever.

Last but not least, I want to thank my parents. Their love and encouragement is

the most important thing for me in these years.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES VII
CHAPTER I: Literature Review 1
Part A Galectins 1
A) Galectin family 1
B) Galectin-1 6
C) Galectin-3 8
Part B Splicing and spliceosomes 12
A) Splicing 12
B) Spliceosome 16
C) Proteomic studies of spliceosomes 19
Part C Galectin involvement in splicing 24
References 29

 

CHAPTER II: Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and

 

 

 

 

 

 

galectin-3 38
Abstract 38
Introduction 39
Materials and Methods 40
Results 43
Discussion 69
References 74

 

CHAPTER III: Dissociation of the splicing and the carbohydrate-binding activities of
galectin-1 76

 

Abstract 76

 

 

 

 

 

 

 

 

 

 

Introduction 77
Experimental procedures 79
Results 83
Discussion 99
References 104
CHAPTER IV: Nuclear galectin containing complexes 106
Introduction 106
Observations 106
Discussion 1 16
CHARPTER V: Conclusion and future plans 119

 

VI

LIST OF FIGURES

 

 

Chapter I
Figure 1: Schematic diagram of the three subfamilies of galectins 2
Figure 2: Schematic diagram depicting the splicing process 13

Figure 3: Depletion and reconstitution of splicing activity of nuclear extract with
galectin-1 and -3 26

 

Chapter 11

Figure 1: Analysis of spliceosomal RNA species immunoprecipitated by various antisera-
All

Figure 2: Time course analysis of spliceosomal RNAs precipitated by anti-gal-l or anti-
gal-3 antiserum 47

 

Figure 3: Immunoprecipitation of 32P-labeled RNA assembled into early (H and E)

 

 

 

complexes 49
Figure 4: Analysis of proteins co-immunoprecipitated by gal-1 antiserum 51
Figure 5: Immunoprecipitation of spliceosomes by anti-Sm antiserum 55

Figure 6: Sequential immunoprecipitation of splicing complexes by gal-1 and gal-3
antisera 58

 

Figure 7: Native gel analysis of splicing substrate incubated with recombinant gal-1,
recombinant gal-3 or NE 62

 

Figure 8: Analysis of spliceosomes immunoprecipitated by anti-gal-l antiserum in

 

 

reactions incubated with GST or GST-ND of gal-3 65
Figure 9: Analysis of spliceosomes precipitated by various antisera at increasing salt
concentrations 67
Chapter III

Figure 1: Characterization of the preparations of fusion proteins containing wild-type
galectin-1 or its N46D mutant by SDS-PAGE 85

 

Figure 2: Comparison of the carbohyrate-binding activity of GST-Gall(WT) and GST-
Ga11(N46D) 88

 

VII

Figure 3: Comparison of the splicing activities of NE, NE after adsorption on ASF-
Sepharose, and depleted NE reconstituted with GST, GST-Gall(WT) and GST-

 

 

 

Gall(N46D) 90
Figure 4: Comparison of the effects of cellobiose, TDG, GST-Gall(WT) and GST-
Gall(N46D) on the splicing activity of NE 93
Figure 5: Comparison of the effects of cellobiose and TDG on the release of Gall from
splicing complexes 97
Chapter IV

Figure 1: Galectin-1 and galectin-3 associate with nuclear complexes which contain
snRNPs and this association is sensitive to ionic strength 108

 

Figure 2: The association of galectins with nuclear complexes is not affected by

 

 

carbohydrate ligands 11 1
Figure 3: Galectin-1 and -3 are mutually exclusive in their association with nuclear
complexes 114
ChapterV

Figure 1: Schematic diagram illustrating the role of galectin-1 and -3 in the splicing
process 120

 

VIII

Chapter 1

Literature Review

Part A Galectins

Galectin family

Galectins are a family of carbohydrate-binding proteins. There are two key
features to characterize these proteins: (i) binding affinity for B—galactosides and (ii)
conserved sequence in their carbohydrate-binding site (1). Galectins have been identified
in many species, including mammal, fish, bird, frog, worm, sponges and fungi (2). Each
member of the galectin family contains at least one carbohydrate recognition domain
(CRD) which has binding affinity for B-galactosides. Several residues in this domain are
critical for saccharide ligand binding, including His49, AsnSl, Arg53, Asn62, Trp69,
Glu72 and Arg74 (using galectin-7 as an example, residues may vary depending on
different galectins).

Fifteen galectins have been discovered and numbered sequentially according to
the accepted numbers for their genes in the Genome Database. According to their
structure, galectins can be separated into three different groups (3) (Figure 1). The first
group is the prototype group. It includes galectin-1,-2, -5, -7, -10, -11, -l3, -l4 and -15.
These proteins contain a single CRD of about 130 amino acids. Crystallography has
shown that the CRD is folded into two anti-parallel B-sheets forming a sandwich like
structure (4, 31). The second group of the galectin family is the chimera type. This group
contains only one member, galectin-3. This protein has a unique amino (N) terminal
domain linked to the CRD. The three dimensional structure of this N-terminal domain is

still unknown. It contains several repeats of Pro-Gly-Ala-Tyr-Pro-Gly-X-X-X, which are

Figure 1: Panel A shows the schematic diagram of the three subfamilies
of galectins (proto-, chimera— and tandem repeat). The prototype is
composed of a single carbohydrate recognition domain (CRD). The
chimera type contains a unique amino terminal domain (key feature is 7-
12 repeats of PGAYPGXXX) and a CRD. The tandem repeat type is
composed of two similar CRDs. Highly conserved residues of the CRD
are shown above the prototype structure. The crystal structure of
galectin-1 CRD is shown in panel B (29).

Figure 1

 

 

 

 

H N R N W E R
Prototype
CRD Galectin-1. -2. -5, -7, -
PGAYPGXXX 10. -11, -13, -14, -15
l lllllll l Chimera
CRD Galectin-3
H Tandem Repeat
CRD CRD Galectin-4, -
N-terminus C-terminus 6‘ '8' '9’ '12

 

conserved in all species. The third group of galectins is the tandem repeat group. The
members of this group (galectin-4, -6, -8, -9, -12) contain two highly similar repeats of
the CRD. These two domains are connected by a short linker peptide (5).

Galectins are believed to be synthesized on free cytoplasmic ribosomes. Although
no signal peptide or conventional nuclear localization sequence has been identified in
galectins, they distribute into all three compartments of the cell—cytoplasm, nucleus and
extracellular space. Some of the galectins bind to membranes and some are in the
extracellular matrix (2).

Different galectins exert different functions. Galectin-2 (14 kD), -4 (36 kD), -6
(33 kD), and -11 (15 kD) are mainly expressed in the gastrointestinal tract of different
animal species (6). They are usually found in the cytoplasm of the epithelial cells. The
functions of these galectins have not been clearly defined. Some evidence suggests that
galectin-11 is involved in the immune responses of the digestion tract (7). Galectin-5 (16
kD) was discovered in rat erythrocytes. It can be found in the cytoplasm and the cell
surface. Galectin-5 can interact with both free saccharides and mammalian glycoproteins
(8). Galectin-7 (15 kD) is a prototype galectin which has a similar folding pattern as
galectin-1. Galectin-7 is mainly expressed in stratified epithelia. Interestingly it is found
predominantly in the nucleus and can be secreted out of the cell (9). Galectin-7 is also a
target for tumor research. It has been suggested to be a pro-apoptotic protein, causing
apoptosis after cell damage. Its tumor suppression function is associated with p53 and the
INK pathway (10). Galectin-8 (35 kD) and -9 (35 kD) are tandem repeat galectins and
both have several isofonns. Galectin-8 is involved in cell adhesion and spreading (11)

while galectin-9 is involved in apoptosis (12). Galectin-9 has also been reported to act as

the uric acid transporter, which is responsible for efflux from urate-producing cells with
renal and gastrointestinal excretion (13). Galectin-10 (also known as Charcot-Leyden
crystal protein) is expressed uniquely in eosinophilic and basophilic leukocytes. Its
sequence only shows moderate similarity to other galectins, while its crystal structure is
highly similar to the prototype galectins (14). Galectin-10 is unique in the galectin family
as it does not bind to B-galactosides, but it does bind to mannose. Galectin-10 has been
found in both the cytoplasm and the nucleus. It has been reported that galectin-10
contains weak lysophospholipase activity (14), but a subsequent study suggests this
conclusion is incorrect. When galectin-10 was depleted by its antibody, the lysates
derived from blood eosinophils retained their full lysophospholipase activity (15).
Galectin-12 (36 kD) is a tandem repeat galectin. It is abundantly expressed in adipose
tissue and mainly concentrated in the nucleus. Galectin-12 was suggested to play a role in
the apoptosis of adipocytes through suppression of cell growth (16). Galectin-13 (16 kD)
was first identified as a soluble human placental tissue protein (17). It has also been
found in adult spleen and bladder, fetal kidney, adenocarcinoma of the liver and
malignant melanoma. Galectin-l4 (17 kD) is found in eosinophils. It has an extended N-
terminal domain beyond the normal CRD of galectins (18). Galectin-15 is a newly
discovered member of the galectin family. Galectin-15 is expressed by the endometrial
luminal epithelium (LE) and superficial ductal glandular epithelium of the ovine uterus.
Galectin-15 has been proposed to have an extracellular role in regulating implantation
and placentation (by functioning as a heterophilic cell adhesion molecule between the
conceptus trophectoderm and endometrial LE). Its intracellular role is to regulate cell

survival, differentiation and function (19).

Galectin-1

Galectin-1 is a member of the prototype galectin family. The molecular weight of
the galectin-l polypeptide is about 14 kD. The N-terminus of galectin-1 is acetylated (20).
The expression and localization of galectin-1 changes according to the stage of
development and differentiation (21, 22). Galectin-1 exhibits dual localization in the cell.
Although largely found in the cytoplasm, galectin-l also exists in the nucleus. In HeLa
cells, treatment with detergent that perrneabilizes the nuclear membrane reveals obvious
nuclear staining of galectin-1 (23). In epidermal Langerhans cells, galectin-1 has been
found in the nucleus by electron microscopy (24). In rat osteoblasts, galectin-1 has been
determined to be a component of the nuclear matrix (25). Galectin-1 is a potent
multifunctional effector that participates in specific protein-carbohydrate and protein-
protein interactions. Many important roles of galectin-1 are exerted through its galectin-
carbohydrate interactions. For example, by interacting with the ganglioside GMl,
galectin-1 is involved in cell growth and neural differentiation (26). Galectin-1 can also
bind its protein ligands through protein-protein interactions, as is the case of Gemin-4 (37)
and H-Ras (27, 28).

The crystal structure of galectin-1 has been determined by X-ray crystallography
(Figure 1). Two identical monomers of galectin-1 assemble into a homodimer. The
overall structure of galectin-1 is a B-sandwich consisting of two anti-parallel fi-sheets of 5
and 6 strands, respectively (29). The N and C termini of galectin-1 are both located at the
dimer interface side and the ligand-binding region is at the far-end (30). This overall

structure is shared by most CRDs of the galectin family. A group of highly conserved

amino acid residues interact with the disaccharide ligands, they are His44, Asn46, Arg48,
Va159, Asn61, Trp68, Glu71 and Arg73 (31).

Many studies have focused on the extracellular functions of galectin-1. In
olfactory neurons, galectin-1 is involved in cell adhesion and establishment and
maintenance of synaptic connectivity (32). In T-lymphocytes, galectin-1 can induce
apoptosis (33). Galectin-1 also interacts with integrin (1781 and through integrin binds to
laminin (34). Recently, its intracellular functions have attracted attention. In the H-Ras
transformed Rat-1 cell, galectin-1 plays an important role in facilitating H-Ras oncogene
product anchoring to the membrane, thus helping the cell transformation process (28).
Galectin-1 directly binds to H-Ras. This interaction has been shown by cross-linking
studies and reciprocal immunoprecipitation using antibodies against both proteins.
Several lines of evidence show this interaction is not related to the carbohydrate binding
activity of galectin-1. First, this interaction is not sensitive to lactose inhibition,
suggesting the interaction is due to protein-protein binding (35). Second, a mutant
galectin-1 has been generated by substituting leucine at position 11 by alanine. This
mutant galectin-1 retains normal carbohydrate-binding capacity but shows diminished H-
Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-
Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast
transformation and PC12-cell neurite outgrth (36). Another protein binding partner
that has been discovered by the yeast two hybrid assay and GST pull down experiment is
Gemin-4 (37). Gemin-4 is a component of the survival of motor neuron (SMN) complex.
The SMN complex is involved in pre-mRNA splicing. Thus the binding of galectin-1 and

Gemin-4 provides evidence to link galectin-1 with splicing.

Galectin-3

Galectin-3 was first discovered as a carbohydrate binding protein, named CBP35
(38). Later, many research groups found this protein independently and named it Mac-2,
IgE binding protein, L-29, LR-29, L-34 and so on. These studies implicate galectin-3 in
many processes in the cell. Unique in the galectin family, galectin-3 is notable by its
unusual N-tenninal domain. The N-terminal domain contains multiple internal sequence
homologies, each of them consists of nine amino acid repeats of Pro-Gly-Ala-Tyr-Pro-
Gly-X-X-X. Differential scanning calorimetry has shown that the N-terminal domain and
the C-terminal domain of mouse galectin-3 have transition temperatures of 40°C and
55°C, respectively (39), suggesting these two domains have different folding structures.
Galectin-3 can form dimers in the cell. The interaction is through both N-terminal domain
and C-terminal domain residues. The C-terminal domain relies on its NWGR motif to
form the dimer, this interaction is saccharide ligand inhibitable and abolished in a W181L
mutation of the galectin-3 polypeptide (40). In the presence of saccharide ligands,
galectin-3 can still form dimers through its N—terminal domain. This has been proved by

cross-linking experiments and site-directed mutagenesis (41 , 42).

Although galectin-3 lacks a conventional signal sequence for transfer into the
endoplasmic reticulum and entry into classical secretory pathway, it does get secreted
into the extracellular space (43). Galectin-3 also shuttles between the nucleus and the
cytoplasm (44). Its intracellular distribution depends on the proliferative state of the cell,
being mainly cytoplasmic in quiescent stage and nuclear in replicating cells (45).
Phosphorylation at N—terminal serine 6 and serine 12 by kinases is necessary for galectin-

3 shuttling from the nucleus to the cytoplasm, because only the phosphorylated form can

be detected in the cytoplasm (46). Phosphorylation is also important for galectin-3’s
activity. Phosphorylation of serine residues inhibits galectin-3 adhesion to the nuclear
matrix (47). On the other hand, phosphorylation favors the anti-apoptotic function of
galectin-3 (48). This has been shown by experiments using site-directed mutagenesis of

galectin-3.

Like galectin-1, galectin-3 also exhibits two different modes of interaction with its
ligands (49). Using lectin-glycoconjugate interactions, extracellular galectin-3 interacts
with the 6-galactoside residues of several extracellular matrix (ECM) and cell surface
glycoproteins via the CRD. In contrast, using both its N-terminal domain and C-terminal
domains, intracellular galectin-3 forms interactions with other proteins via protein-protein
associations. One example of such interactions is galectin-3 with TTF -1. GST pull-down
assays demonstrated a direct interaction between galectin-3 and the homeodomain of

TTF-l (50). These binding properties could enable galectin-3 to exert multiple functions

(51).

Intracellularly, galectin-3 is involved in regulating the cell cycle. It can arrest cells
in G1 or at the G2/M phase. The basic regulating steps are downregulating cyclins A and
E, upregulating p21 and p27 cyclin inhibitors, and hypophosphorylating Rb protein (52).
Galectin-3 is also involved in cell proliferation and cell death. In 3T3 fibroblasts,
galectin-3 can stimulate cell proliferation by its upregulation and altered pattern of
distribution and phosphorylation (53). The mitogenic potential of galectin-3 has been
demonstrated in several experiments. In human lung fibroblasts, cell proliferation is

associated with the expression of galectin-3 (54). In human leukemia T-cells, when

transfected with galectin-3 cDNA, the cells show higher growth rates than control
transfectants (55). In addition to regulating cell proliferation, galectin-3 favors cell
survival by protecting from apoptosis induced by a variety of death signals and anoikis
(52, 56). Galectin-3’s anti-apoptotic activity seems to be related to the highly conserved
NWGR motif sequence. Galectin-3’s association with bcl-2 (56) and x/ALG-2-interacting
protein 1 (the human homologue of ALG-2 linked protein) (57) is also important to this
activity. By its pro-proliferative and anti-apoptotic action, galectin-3 is considered as an
immediate early gene possibly implicated in tumor growth. This effect has been shown by

the abnormal expression of galectin-3 in several neoplasms (58).

Extracellularly, galectin-3 is involved in regulating cell adhesion. Cell surface
galectin-3 binds to a serum glycoprotein, serving as a cross-linking bridge between
adjacent cells and promotes homo and heterotypic cell-to-cell interactions (59). On the
other hand, galectin-3 associates with the alBl-integrin receptor, downregulating cell
adhesion to the ECM component laminin, thus producing an anti-adhesive effect (60). In
the process of tumor invasive and metastasis, these types of adhesion changes play a very
important role. Presumably, galectin-3 might increase the interactions among tumor cells
and the interaction of tumor cells with the endothelium; it also could alter the adhesion of
cells to the basement membrane glycoprotein laminin. Under certain circumstances,
galectin-3 may promote cell adhesion to laminin, as shown in neutrophils (61). This effect
is believed to play a role in the traversing of neutrophils through the basement membrane

at the site of inflammation.

10

Another important function of extracellular galectin-3 is the modulation of
inflammation and allergy. By binding to IgE and the IgE receptor, galectin-3 can induce
activation of mast cells and basophils, which is a central event in allergy (62). Galectin-3
also helps eosinophils and neutrophils bind IgE, therefore playing an important role in
mediating their IgE-dependent effector function (51). Finally, galectin-3 is reported to be
capable of binding, internalizing, and degrading AGE (advanced glycation end products),

thus playing a role in diabetes and aging (63).

11

Part B Splicing and spliceosomes

Splicing

Pre-mRNA splicing is a process which removes introns from the pre-mRNA and
ligates exons together to form the mature messenger RNA (64). Two transesterification
reactions take place in this process. In step 1, the 2’-OH group of the adenosine at the
branch point attacks the phosphate group of the 5’ splice junction (guanosine) forming
exon-l and lariat-exon-2 intermediates. In step 2, the free 3’-OH of exon-l attacks the
phosphate group of the 3’ splice junction (guanosine), resulting in ligation of the exons
and release of a free lariat which is degraded later (65) (Figure 2). Several regions on the
pre-mRNA are essential for these reactions. The 5’ splice junction has the consensus
sequence AGzGU. At the 3’ splice junction the consensus sequence is AG in a context of
semi-conserved CAGzG. A branch point region is also crucial (66). It is usually 30
nucleotides (nt) upstream of the 3’ splice site. In yeast, the branch point sequence is an
absolutely conserved sequence of UACUA(A)C. In vertebrates, it is variable with
PyNPyPu(A)Py. Besides these regions, mammals also contain a polypyrimidine stretch
upstream of the 3’ splice junction to facilitate recognition.

Pre-mRNA splicing is an important post-transcriptional process in eukaryotic
cells. This process is accomplished by a large macromolecular complex called the
spliceosome. The spliceosome contains five small nuclear ribonucleoprotein complexes
(snRNPs) and a large number of non-snRNP proteins (many are arginine/serine (RS)
domain containing proteins). The snRNPs involved in splicing process are U1, U2, U4,
U5 and U6 snRNP (67). There are a few exceptions in the splicing process. For example,

group 2 introns can splice in the absence of proteins (68). But these occasions are rare.

12

Figure 2: Schematic diagram depicting the splicing process. In
step 1, the 2’-OH group (A) branch point attacks the phosphate
group of the 5’ splice junction (guanosine) forming exon-l and
lariat-exon-Z intermediates. In step 2, the free 3’-OH of exon-l
attacks the phosphate group of the 3’ splice junction (guanosine),
resulting in ligation of the exons and a free lariat which is
degraded later (105).

13

Figure 2

5. exon intron 3' exon
film
stifoflofio 'U

 

 

 

 

 

5' :ma’H— OH 3'

 
 

 

 

Spliced exons

 

14

The snRNPs contain a single low molecular weight RNA and several protein
components. The snRNAs are about 200 nucleotides in length. Their 5’ end form a 2,2,7-
trimethyl-guanosine cap. There are six common sets of snRNP core proteins in this
particle in addition to one or more snRNP-specific proteins (67). Besides functioning in
pre-mRNA splicing, snRNPs are also involved in 3’ end processing of pre-mRNAs (69).
The key component in the snRNPs is the snRNA. The snRNAs can base pair with pre-
mRNA in the conserved sequence motif and allow the snRNP particles to initiate the
assembly of the spliceosome (70).

The cellular amount of snRNPs is variable according to species. In mammalian
cells, the snRNAs are rather abundant. There are about 1x105 to 1x106 copies in each
human cell. In yeast, there are about 10 times less.

Except for U6, U snRNAs are transcribed by Pol 11 (DNA dependent RNA
polymerase II) in the nucleus. They are then capped with a 7-methyl—guanosine and
exported to the cytoplasm. In the cytoplasm, a set of Sm proteins is assembled onto the
snRNAs, the 5’ end of snRNAs is also hypermethylated, forming the 2,2,7-trimethyl-
guanosine cap structure. The cap structure and the Sm protein ring serve as nuclear
import signals, bringing the snRNPs back into the nucleus. In the nucleus, snRNPs first
appear in the Cajal Body. In this nuclear structure, snRNPs undergo further assembly.
Several snRNP-specific proteins are added, bases of the snRNA are modified, and
U4/U6.U5 tri-snRNP is assembled (71, 72, 73). U6 goes through a different pathway. It is
transcribed by Pol III and does not get exported to the cytoplasm. Thus, U6 only has a

mono-methyl cap and remains in the nucleus. Since U6 does not enter the cytoplasm, it

15

does not acquire the Sm proteins. Instead, eight Sm-like (LSm) proteins replace the Sm
proteins and form the core proteins (72, 74).

The snRNPs carry out essential functions during the splicing reaction. Via the
interaction of their RNA and protein components with the pre-mRNA, they mediate the
recognition and subsequent base pairing of the 5' and 3' splice sites of an intron. In
addition, they form the three-dimensional structure which is required for forming the
spliceosomes‘ two active sites. Components of the snRNPs also appear to catalyze the
two transesterification reactions that occur during splicing (74).

Spliceosome

Newly transcribed pre-mRNA is covered by hnRNP (heterogeneous nuclear
ribonucleoprotein) proteins, forming the initial H complex. This is a non-specific
complex and not all H complexes can be chased into later splicing complexes.
Spliceosome assembly is a process in which the 5 snRNPs and many non-snRNP proteins
orderly assemble onto the pre-mRNA. The conventional model for spliceosome assembly
is a step-wise process (75, 76). The first step is the assembly of U1 snRNP onto the 5’
splicing site of the pre-mRNA. The consensus sequence at the 5’ splice junction is
recognized by U1 through an RNA-RNA complementary base pairing (77). The second
step is the addition of U2 snRNP. A U2 snRNP specific protein (U2AF) plays an
important role in this step. U2AF has two sub-domains. The small subunit is about 35 kD,
called U2AF35; the large one is 65 kD, named U2AF65. U2AF35 recognizes the 3’
splicing site with the sequence of AG and binds to this region, while U2AF65 binds to
the polypyrimidine tract (78). The binding of U2AF recruits the U2 snRNP and does not

' require ATP hydrolysis. With U1 snRNP and U2AF bound to pre-mRNA, a new splicing

16

complex (E complex) forms (79). B complex is also called the commitment complex.
This is the real starting point of the functional spliceosome. Given the right conditions,
all of the E complexes can be chased into higher ordered complexes. There are also other
important proteins needed to assemble onto the RNA before U2 snRNP can bind. SF 1
(called BBP in yeast) recognizes the branch point A and is critical in facilitating the
assembly of U2 snRNP. SF2 and SC35 bring the 5’ and 3’ splice sites in proximity by
interacting with U2AF35 and thus change the spatial structure of the complex (80, 81).
Although U2 snRNP has been reported to associate with the B complex, it is widely
believed that the binding of U2 snRNP to the branch point forms the next higher ordered
complex, the A complex. This is accomplished by the U2 snRNA replacing the SF 1
protein and base pairing with pre-mRNA at the branch A region using its 5’ end. This
switch of protein-RNA interaction to RNA-RNA interaction is the first ATP dependent
step in spliceosome assembly and starts the formation of the catalytic complex (80).

After A complex is formed, U4/U6.U5 tri-snRNP comes into play. In this tri-
snRNP, U4 and U6 associate through snRNA-snRNA interactions while US snRNP binds
to them through protein-protein interactions. The binding of this tri-snRNP with the pre-
mRNA forms an intermediate complex (B complex) before the spliceosome becomes
splicing active (76, 82). Before U6 snRNA can bind to the complementary region on the
pre-mRNA, the base pair between U4 and U6 needs to be disrupted. This process is
accomplished by several DExD/H box proteins (84). Prp44, also called Brr2, has been
implicated in unwinding the U4/U6 helices and likely confers the ATPase activity (85).
U4 snRNP protein Prp28 and U6 snRNP protein Prp24 have also been proposed to

directly or indirectly influence U4fU6 unwinding (86). A US snRNP protein Prp8 has

17

been suggested to play an important role in regulating the activity of the DExD/H-box
proteins (87). Overall this unwinding is an ATP-dependent process and requires multiple
protein involvement. After the U6 snRNA dissociates from the U4 base pair, it displaces
U1 RNA from the 5' splice site, establishing a new pairing between U6 RNA and the 5'
splice site. Following this event, U1 and U4 snRNP are released from the spliceosome.

Accompanying these component changes, the splicing complex also goes through
a conformation rearrangement. When U2 and U6 snRNA base pair together, the catalytic
core is formed and the active splicing complex (C complex) finally forms (76). These
conformational changes appear to require the branchpoint sequence, presumably because
the activated U2 snRNP has a role in the process. The role of U5 snRNP is mainly
contributed by its protein components. U1-70K interacts with the U5 snRNP proteins
Prp44/Brr2 and Prp8 (85, 88) and with protein Ex084. Ex084, in turn, interacts with Prp8
(89). Prp8 can also interacts with U1 snRNP proteins Prp39 and Prp40 (87). In fact, a
Ul/U 5 bi-snRNP has been identified in yeast cell extracts (90), which further strengthens
these discoveries.

Recently, a new model for the assembly of spliceosome has been proposed by
Stevens et a1. They believe that in the nucleus there is a massive ribonucleoprotein
complex, containing five U snRNPs (U1, U2, U4 U5 and U6), called penta—snRNP (91).
The facts which support that such a complex exists are: a) the snRNPs appear to be in
stoichiometic proportions and b) the proteins found in this purification are specific. This
complex contains all the snRNP proteins and 13 known splicing associated proteins. In a
functional study, Stevens et a1. (91) have shown evidence that the penta-snRNP complex

is significant for pre-mRNA splicing. Extracts depleted of endogenous snRNAs by

18

nuclease digestion can be restored of their splicing activity by adding back the isolated
penta-snRNP. Moreover, when they used tagged snRNAs for a mixing experiment, they
found that during spliceosome assembly the components of the snRNPs do not exchange
with each other, which strongly supports the idea that the penta-snRNP works as a pre-
existing unit for splicing. Stevens et al. also claimed that the difference between this
model and the traditional one may be due to different isolation conditions (91). Although
no other significant developments have been reported recently, this model is still
considered as an innovative point of view toward spliceosome assembly.

Besides the major splicing complexes, there is a minor class of introns using
spliceosomes containing U11, U12 and U5 snRNPs. Unlike the GT-AG rule of the vast
majority of introns, this minor class of introns contains AT and AC at their 5’ and 3’ ends,
respectively. In this spliceosome, U11 and U12 resemble the U1 and U2 roles in the
major spliceosome, respectively. US also fimctions in this spliceosome, but there is no
evidence to suggest the involvement of U4 or U6 snRNPs (92, 93, 94).

As has been stated, besides snRNPs, there are many other components in the
spliceosome. Their roles in splicing have not been completely studied. It is generally
believed that they facilitate the assembly of the snRNPs and stabilize the spliceosome as
an active complex. In recent years, using proteomic studies, many of the spliceosome
components have been determined and analyzed.

Proteomic studies of spliceosomes

For a long time, it has been known that spliceosomes are large and complicated

complexes. In early studies, researchers used depletion and reconstitution and budding

yeast genetic analysis to identify the components of these complexes. But until recent

19

years, no one has shown a complete picture of the spliceosome. In the past decade, a new
method has been used to analyze the spliceosome. It is tandem mass spectrometry. The
first splicing complexes analyzed by mass spectrometric methods were the hnRNP
complex assembled on mammalian pre-mRNA (95) and subunits of the yeast
spliceosome (96, 97). Subsequently, large-scale analysis of human multi-protein
complexes on in vitro assembled spliceosomes was performed. The first step of such
analysis is to isolate the spliceosome from the cell nuclear extract. Different ways of
purification have been exploited.

In 1999, Fischer et a1. (98) used antibodies against the trimethylguanosine (m3G)-
cap to immunoaffinity-purify snRNPs from HeLa nuclear extracts and separated the
complexes on 10 to 30% glycerol gradients. Fractions containing different snRNP
complexes were pooled and selected with biotinylated 2'-O-methyl oligonucleotides and
streptavidin agarose. Thus, these studies provided a thorough analysis of the snRNP
complexes.

In 2002, using a similar method, Rappsilber et al. (99) assembled a mixture of
spliceosomal complexes on biotinylated, radioactively labeled RNA from adenovirus
(ADI) and B-globin (AL4) transcripts. Samples were subjected to gel filtration and
affinity selection using streptavidin beads. The peptides bound to the high-performance
liquid chromatography (HPLC) column were analyzed.

In another proteomic study, Zhou et al. (100) also used two distinct pre-mRNAs
(adenovirus major late, AdML, and Fushi tarazu, th) to form a mixture of all stages of
spliceosomes, including complexes that had undergone the first or second catalytic steps

of the splicing reaction. In this study, they used a different purification approach. The

20

substrate pre-mRNA contained three hairpin structures which bind to the M82
bacteriophage coat protein. Using an MSZ-MBP fusion protein (MBP is maltose-binding

protein), these complexes could be used for affinity purification.

With the purification of different stage spliceosomes, the next step was to subject
them to mass spectrometric analysis. Mass spectrometric methods and human sequence
databases have continued to improve in recent years, allowing both increased sensitivity
and higher throughput. It is now possible to analyze mixtures of hundreds or even
thousands of peptides by liquid chromatography coupled to tandem mass spectrometry
(LC MS/MS). Improved software and databases containing most human genes are also
now available, allowing automated data processing of the large volume of acquired mass

spectra (101 ).

These proteomic studies have shown a large number of protein components of the
spliceosomes. In Reed’s study, a total of 145 distinct proteins were identified as shared
between the two types of pre-mRNA spliceosomes, including 88 known splicing
factors/snRNP proteins/spliceosomal proteins (100). Among these 88 known proteins, 41
are distinct proteins found in U1, U2, U4, U5 and U6 snRNPs. The other 47 proteins
include members of the SR family of splicing factors, DExD box splicing factors,
proteins that are found in the salt-stable spliceosome 'core', second catalytic step splicing
factors and known components of the spliced messenger ribonucleoprotein (mRNP) that
promote mRNA transport to the cytoplasm. Interestingly, a number of factors involved in
genetic diseases, like retinitis pigmentosa (U4/U6-61K, U4/U6-90K, U5-220K) or cancer

(WTAP), are also associated with spliceosomes. Besides these known splicing factors,

21

they also found 58 proteins that have not been previously identified as spliceosomal
proteins in humans. 36 of these proteins have previously been named and the rest are
uncharacterized. Some of these 58 proteins have been suggested by others to be involved
in splicing. One example is cyclophilin-like proteins. Recent studies revealed a role for
one cyclophilin (U4/U6-20K) in splicing, and these data suggest that cyclophilins may
have a general function in mediating conformational changes in the spliceosome. The fact
that some of these proteins contain domains which resemble RNA recognition motifs or
DExD boxes, are known to co-localize with splicing factors in nuclear speckles and/or to
localize in the nucleus and many of the proteins have known yeast counterparts that

function in mRNA processing strongly surpport they are also splicing related proteins.

Mann’s study also revealed the same type of protein catalog of the spliceosomes,
only with a larger number of components (99). A total of 311 proteins were identified.
More than 40 percent of the identified proteins have a known function related to splicing
or RNA processing. The first group is the core snRNP proteins (Sm proteins), all that are
present in U1, U2, U4, and U5 snRNPs were identified; six of the seven Lsm proteins that
substitute for Sm proteins in the U6 snRNP were also detected. The second group is
39 non-snRNP protein splicing factors, including early-acting factors such as splicing
factor 1 (SF1) and late factors such as Slu7(required for the second catalytic step of the
splicing reaction) and Aly (for RNA export). The third group is 20 hnRNP proteins which
are defined by a common RNA-binding motif and are thought to have diverse functions
in RNA protection and processing. Some hnRNPs are known to be splicing factors, such
as GRY-RBP/hnRNP Q. The fourth group is 27 other RNA-processing-related proteins.

These proteins have functions closely associated with splicing, such as 5' cap binding

22

proteins (CBP) 20, CBP 80 and the 3’ end cleavage and polyadenylation factor (CPSF).
This research team also found a large number of unknown novel proteins in their analysis.
Compared to their previous study, they found 96 new proteins associated with
spliceosomes. Many of these proteins have a domain structure suggesting their role in
splicing/RNA processing -- 55 proteins have sequences similar to known splicing factors
or domains that implicate them in RNA processing. They also found a number of proteins
involved in transcription and cellular regulatory mechanisms, indicating some form of

coupling of these processes to splicing.

Although proteomic analysis is a powerful tool to analyze spliceosome, it is
important to notice that not all the spliceosome components would be detected. If a
protein was small or not abundant, it would generate few detectable tryptic peptides, and
thus not be able to be detected by this analysis. Moreover, since most of the proteomic
studies use very stringent conditions for complex purification, if a component is not
tightly associated with the spliceosome, it is likely this protein will be lost during the

purification step.

23

Part C Galectin involvement in splicing

Galectins have been known for many years. But the intracellular function of
galectins has only been recognized in recent years. The splicing activity of galectin-1 and
galectin-3 was first hinted by cesium sulfate density gradient centrifugation. In this
experiment, galectin-3 was found not only as a free protein, but also co-sedimenting with
hnRNP and snRNP complexes, which are related to pre-mRNA splicing. In another
experiment, when treated with ribonuclease, the immunoflurescence staining pattern of
galectin-3 associated with the nuclear structures was abolished, indicating galectin-3 is
associated with ribonucleoprotein complexes. The third line of evidence is that confocal
immunofluorescence microscopy shows galectin-3 and galectin-1 co-localize with Sm
core proteins and SC35, a non—snRNP splicing factor, in a subnuclear region called
speckles. Speckles are the storage sites forvsplicing factors (23, 103). All of these results

point to the possibility that galectin-3 is a splicing factor.

To confirm galectins as splicing factors, functional assays have been performed.
Two types of assays were employed. The first assay is using denaturing gel
electrophoresis to analyze spliceosomal substrate RNA species. During the splicing
reaction, pre-mRNA will be converted into mature mRNA. Together with intermediate
RNA species, they will be resolved by this gel system (102). The second way of analysis
is to form different splicing complexes and resolve these complexes by native gel
electrophoresis. An ideal assay will show H/E, A, B and C complexes (102). The role of
galectins in splicing can thus be illustrated by saccharide inhibition and depletion-

reconstitution experiments.

24

HeLa cell nuclear extract (NE) is capable of carrying out in vitro splicing
reactions. When saccharides with high galectin binding affinity (lactose or TDG) were
added, the in vitro splicing reaction was inhibited. This phenomenon was not observed
when saccharides without affinity for galectins were added. This suggests a role of
galectins in splicing. In the process of depletion and reconstitution, removal of galectins
from NE, either by lactose affinity chromatography or by antibody-mediated depletion,
abolished the splicing activity of the nuclear extract. When galectin-l or galectin-3 was
added to the depleted extract, the splicing activity was restored (Figure 3), confirming
galectin-l and -3 are bona fide splicing factors and their roles in splicing are redundant

(103).

Recently, the discovery of a galectin-1 protein ligand gives support for the above
conclusion. Using a yeast two hybrid analysis, a binding partner of galectin-1, Gemin—4,
was isolated from a HeLa cell cDNA library. The direct interaction between galectin-l
and Gemin-4 was also confirmed by a GST pull down experiment (37). Gemin-4 is a
component of the SMN complex. The SMN complex is responsible for the assembly of
snRNPs in the cytoplasm and the recycling of snRNPs in the nucleus after one round of
splicing (104). Thus, a link between galectin-1/—3 and snRNP complexes was established.
The most recent discovery in this area is the association of nuclear galectins with a
transcription factor TF II-I. Since pre-mRNA splicing appears to occur both co-
transcriptionally and post-transcriptionally, this observation may suggest a linkage role of

galectins in mRNA processing.

25

Figure 3: Depletion and reconstitution of splicing activity of nuclear
extract with galectin-l and -3. Nuclear extract lost its splicing activity
through antibody-mediated depletion (lane 2). When recombinant galectin-
] or galectin-3 were added back (lanes 3 and 5), the splicing activity of the
depleted nuclear extract was restored. This reconstitution was inhibitable
by thiodigalactoside (TDG) (lanes 4 and 6) (103).

26

Figure 3

UB of agall and agal3

I

(3

B e [e
< + +
+ 9mg...
(6 .—

Li’ EDEDPPED

    

MM Ilvlilv‘li 'W'
will

w m w-

27

Although these results have suggested that galectins are splicing factors, it must
be acknowleged that mutant mice with galectin-1 and galectin-3 genes knocked out are
viable and fertile (though some physiological changes could be observed). This indicates

that galectins’ function could be replaced by other proteins in a living system.

28

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37

Chapter 2

Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1

and galectin-3

Nucleic Acids Res. 2006 Oct 6; Epub
Abstract
We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors.
Now we provide evidence that both galectins are directly associated with spliceosomes
by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific
antisera.- Both galectin antisera co-precipitated splicing substrate, splicing intermediates
and products in active spliceosomes. Protein factors co-precipitated by the galectin
antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early
spliceosomal complexes were also immunoprecipitated by these antisera. When splicing
reactions were sequentially immunoprecipitated with galectin antisera, we found that
galectin-l-containing spliceosomes did not contain galectin-3 and vice versa, providing
an explanation for the functional redundancy of nuclear galectins in splicing. The
association of galectins with spliceosomes was (i) not due to a direct interaction of
galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had
only a minimal effect on snRNP association. Finally, addition of excess amino terminal
domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes,

explaining the dominant negative effect of the amino domain on splicing activity. We

38

conclude that galectins are directly associated with splicing complexes throughout the
splicing pathway in a mutually exclusive manner and they bind a common splicing
partner through weak protein-protein interactions.
Introduction

Before transport to the cytoplasm for translation, RNA transcripts in the form of
pre-messenger RNA (pre-mRNA) assemble into a macromolecular structure termed the
spliceosome. During subsequent remodeling events of spliceosomes, introns are removed
and exons are ligated to form mature mRNA. In vitro cell-free assays using simple
splicing substrates and nuclear extracts (NE) have established the basic sequence of
biochemical events and ordered series of complexes in the pathway (1-4). In this dynamic,
multi—step process, over 300 proteins and five ribonucleoprotein particles (snRNPs)
orchestrate two trans-esterification reactions that result in the formation of mRNA (5-9).
We previously identified two polypeptides in NE from HeLa cells (galectin-l and
galectin-3) required for splicing (10, 11). Depletion of both galectins from NE, either by
lactose-agarose affinity or double antibody adsorption chromatography, abolished
splicing activity and halted spliceosome assembly at an early step. Addition of either
galectin restored both splicing activity and spliceosome formation. These data suggested
that galectins are, indeed, splicing factors and that they are functionally redundant.

Galectins are a family of carbohydrate-binding proteins with specificity for
galactose or galactose-containing glycoconjugates that localize both intracellularly and
extracellularly ( 12). Two of the galectins (galectins-1 and —3) are ubiquitous in
mammalian tissues and have been documented in the nucleus and cytosol. Galectin-1

(gal-1) is composed of a single carbohydrate recognition domain (CRD) of ~130 amino

39

acids. Galectin-3 (gal-3) has an amino terminal domain (ND) of ~130 amino acids
containing multiple repeats of the amino acid sequence PGAYPGXXX of unknown
function fused to a CRD whose sequence is conserved when compared to the CRD of
gal-1 and the other galectins. As we investigated the role of gal-1 and gal-3 in splicing,
we noted several key differences. First, recombinant gal-3 is ~5-10 times more efficient
at reconstitution of splicing than gal-1 or the isolated CRD of gal-3 (11). Second,
although both galectins co-localize with known splicing factors (i.e., the Sm core
polypeptides of snRNPs and SC35) and each other in nuclear speckles, there are regions
of non-overlap (13). Lastly, the isolated ND of gal-3 inhibits splicing chemistry and
spliceosome formation in a dominant negative manner when added to splicing competent
NE (14).

Although these data are consistent with galectins being splicing factors, they do
not address whether their role in splicing is direct or indirect. In this study, we provide
evidence for a direct role of galectins in the splicing pathway by showing that galectin-
specific antibodies co-immunoprecipitate pre-mRNA, splicing intermediates and products
throughout the entire splicing pathway. We have investigated various characteristics of
galectin incorporation into spliceosomes and conclude they interact via weak protein-

protein interactions with another spliceosomal component.

Materials and Methods

Antibodies

Polyclonal rabbit antiserum against recombinant rat gal-1 was a gift from Doug Cooper

(University of California, San Francisco) (14). Additionally, we raised a second

40

polyclonal rabbit antiserum against recombinant human gal-1 expressed as a fusion
protein with glutathione S-transferase (GST). A human gal-1 cDNA clone was kindly
provided by Jun Hirabayashi (15). The gal-1 cDNA insert was isolated after BamHl
digestion and inserted into pGEX-ZT. Following expression in DHSa, the fusion protein
was purified by glutathione-agarose affinity chromatography and used to immunize
rabbits. Several polyclonal rabbit antisera to murine gal-3 were produced as described in
Agrwal et al. (16). When used in immunoprecipitation experiments (Figures 4 and 6),
each galectin antiserum was mono-specific for a galectin (i.e., anti-gal-l precipitated only
gal-1 from splicing reactions and anti-gal-3 precipitated only gal-3). Human autoimmune
serum (ENA anti-Sm) was purchased from The Binding Site. Anti-Slu7 antibodies were
purchased from Santa Cruz Biotechnology and anti—Ran antibodies from BD Biosciences.
A monoclonal antibody against hnRNP C1/C2 (4F4) was provided by Gideon Dreyfuss
(University of Pennsylvania). For immunoprecipitation experiments, all antibodies were
covalently cross-linked to protein G-Sepharose fast flow 4B beads (Sigma) as previously

described (11, 14) generally using a 2:1 ratio of antiserum to protein G beads.

Co-immunoprecipitation of 32P-labeled-MINX RNAs

Splicing reactions containing 60% by vol. HeLa NE were assembled with 32P-
MINX RNA without or with ATP (1.5 mM) and creatine phosphate (20 mM) and
incubated at 30°C for the times indicated as previously described (10, 11, 14). HeLa cells
were obtained from the National Cell Culture Center. Typically 60—100 111 of the splicing
reaction mixture was diluted to 0.5 ml with 60% buffer D (D60) and incubated with 30-
50 ul antibody cross-linked protein G-Sepharose beads at 4°C for l - 2 hr. After washing

with D60 containing 0.05% Triton X-100 (three washes, each with 1 ml buffer), the

41

bound material was eluted with 20 p1 of SDS-PAGE sample buffer. The eluted sample
was divided into two aliquots. RNA was purified from one by incubating at 37°C for 20
min with proteinase K (4 mg/ml final concentration) and diluting to 100 pl with 125 mM
Tris (pH 8), 1 mM EDTA, 300 mM sodium acetate. RNA was extracted with 200 pl of
phenol-chloroforrn (50:50 [vol/vol]), followed by 200 pl of chloroform. RNA was
precipitated with 300 pl of ethanol at —80°C. The extracted RNA was dissolved in 10 pl
of sample buffer (9:1 / fonnamide: bromophenol blue) and subjected to electrophoresis
through 13% polyacrylamide (bisacrylamide-acrylamide 1.9:50 [wt/wt])-8.3 M urea gels.
The radioactive RNA species were revealed by autoradiography and quantitated by
phosphorimage analysis (Molecular Dynamics). The other aliquot was subjected to
electrophoresis in 12.5% or 15% SDS-PAGE (bisacrylamide-acrylamide 0.9:30 [wt/wt])
and analyzed by western blotting.

The salt sensitivity of the association of galectins and snRNPs with spliceosomes
was determined as follows. Active splicing complexes (incubated for 60 min with ATP at
30°C) were incubated with antibody-coupled beads. Following the initial binding, the
beads were washed either with 60, 130 or 250 mM KCl-containing buffer and the bound
material was eluted with SDS-PAGE sample buffer. The RNA was extracted and

analyzed by denaturing gel electrophoresis as described above.

Native gel electrophoresis
To evaluate binding of the galectins to the splicing substrate, recombinant gal-1 or
recombinant gal-3 (expressed in DHSa and purified by lactose-agarose affinity

chromatography), or NE was incubated with 32P-MINX under splicing conditions.

42

Following incubation, the reactions were subjected to native gel electrophoresis (4%
polyacrylamide gels [bisacrylamidezacrylamide 1:80 wt/wt]) (17) and complex formation
revealed by autoradiography. Agarose native gel electrophoresis to identify H or E

complexes was performed as described by Das et a1. (18) followed by autoradiography.

Results

Galectins-1 and —3 are associated with spliceosomes

Previously we showed that depletion of gal-1 and gal-3 from HeLa NE abolished
pre-mRNA splicing and arrested spliceosomes before formation of active complexes (i.e.,
only early complexes formed) (10). Addition of either recombinant gal-1 or gal-3 to the
galectin-depleted NE resulted in reconstitution of splicing activity and active spliceosome
formation (10, 11). We concluded that these two nuclear galectins are redundant splicing
factors. However, there was little information to suggest whether their involvement in
the splicing pathway was direct or indirect.

To test for a direct association of galectins with spliceosomes, we determined
whether radiolabeled splicing substrate in splicing complexes could be precipitated by
galectin-specific antisera. As seen in Figure 1 (lane 1), after incubation in HeLa NE with
ATP for 60 min, the 32P-MINX splicing substrate is converted to processing
intermediates and products. Typically, 20-35 % of the RNA detected following 60 min is
ligated product. In the absence of ATP, no processing of the MINX substrate occurs (see
below). When splicing reactions that had been incubated for 60 min are

immunoprecipitated by galectin specific antisera, all species of RNA, the pre-mRNA

43

Figure 1: Analysis of spliceosomal RNA species immunoprecipitated by
various antisera. Equal aliquots of splicing reactions incubated for 60 min
with 32P-MINX and ATP were subjected to antibody adsorption and the
bound RNA analyzed by denaturing gel electrophoresis and
autoradiography. Lane 1, RNA in the splicing reaction subjected to
immunoprecipitation; lane 2, RNA bound to pre-immune serum; lane 3,
RNA bound to anti-Sm; lane 4, RNA bound to anti-gal-l and lane 5, RNA
bound to anti-gal-3. The positions of the various RNA species are
diagrammed on the right in this and subsequent figures analyzing
spliceosomal RNAs.

44

Figure 1

’5 all};
Eases

 

 

Milli“.

”Milt

 

 

 

45

substrate, the intermediates and the mature products, are found in the precipitates (lanes 4
and 5). In contrast, pre-immune serum did not precipitate significant quantities of labeled
RNA (lane 2). Human autoimmune serum reactive against the Sm polypeptides of the
snRNPs precipitated all species of radiolabeled spliceosomal-associated MINX as
expected (lane 3). Thus, gal-1 and gal-3, like Sm proteins, are associated with active
spliceosomes.

To characterize in greater detail galectin-containing splicing complexes, splicing
reactions were subjected to antiserum selection during a time course experiment and
RNAs (Figures 2 and 3) and some of the proteins (Figure 4) in the immunoprecipitates
were characterized. Each galectin antisera co-immunoprecipitated the splicing substrate
throughout the time course of the splicing reaction. At the earliest times sampled, both
antisera precipitated MINX pre-mRNA, most probably in the form of H/E complexes
(see below). Splicing intermediates and ligated exons were precipitated as they appeared
during the kinetic analysis (Figure 2, beginning at 20 min for the anti-gal-l time course
and at 40 min for the anti-gal-3 time course). An internal control for the specificity of
RNA precipitation is apparent in these analyses. Degraded RNAs observed at 0, 5 and 10
min (highlighted by arrows to the left of the input lanes in Figure 2) are not detected in
the immunoprecipitated complexes at these times. Both galectin antisera appeared to
immunoprecipitate the excised lariat RNA species preferentially (compare the ratios of
lariat to pre-mRNA species in the precipitates and in the input at the 40 and 60 min time
points in Figure 2). Both observations (i.e., no precipitation of degraded RNAs and
preferential precipitation of free lariat) argue against non-specific adsorption of

radioactive RNA species to beads since the precipitated RNAs do not reflect the same

46

Figure 2: Time course analysis of spliceosomal RNAs precipitated by anti-
gal-l or anti-gal-3 antiserum. 32P-MINX was incubated with ATP in NE at
30°C. At the times indicated, the reactions were subjected to galectin affinity
adsorption and the bound fraction separated into two parts. RNA was
extracted from one portion and analyzed by denaturing gel electrophoresis.
MINX RNA in splicing reactions at 0, 5, 10, 20, 40 and 60 min (input) and
MINX RNA precipitated by anti-gal-l or anti-gal-3 antiserum. Arrows at left
indicate degraded RNA species in the input material.

47

Figure 2

Input aGal—1
Min 0 510 20 4060 0 510 204060

 

     
 

2.“.31tlim— 111:2:

' i ‘ .1 l
K ‘
W” ."’""‘ mm "Ml Mm .... m
it” ‘ éinllnl

Input aGal—3

 

48

Figure 3: Immunoprecipitation of 32P—labeled RNA assembled into early
(H and E) complexes. Panel A: MINX RNA was incubated with NE at
30°C in the absence of ATP for the indicated times. The reaction mixtures
were loaded directly onto 1.5% agarose gels. Following electrophoresis,
autoradiography was performed to detect RNA-containing complexes.
Panel B: Splicing reactions containing MINX RNA in the absence of ATP
at 0, 20, 40 and 60 min incubation at 30°C (lanes 1-4) were incubated with
anti-gal-l immobilized and the bound material subjected to analysis by
denaturing gel electrophoresis (lanes 5-8).

49

Figure 3

NE minus ATP
0 20 40

« Input aGal-1

0204060 020 40 60
E ' ,

 

50

Figure 4: Analysis of proteins co-immunoprecipitated by gal-1
antiserum. HeLa NE (NE) and bound fractions from anti-gal-l
immunoprecipitates (0G1) were blotted for Sm B/B’, hnRNP C1/C2,
Slu7, gal-1 and gal-3.

51

Figure 4

NE aGal1
Sm BIB, W .4411»:er .

Gal-1 E29”
hnRNP c1/c2 W

6“” W

    
 
 

52

relative amounts of the different RNA species in the sample used for immunoselection
(input).

Detecting MINX pre-mRNA in galectin immunoprecipitates at early times in the
splicing reaction prompted us to investigate the association of galectins with complexes
characterized as H or E splicing complexes. H complexes form upon addition of a
substrate to a splicing extract even when incubated on ice. Following incubation at
elevated temperatures, H complexes are converted to E (early) complexes that are the
immediate precursors of active spliceosomes. Neither H nor B complex assembly requires
ATP (18). To test for galectin association with these two complexes, MINX pre-mRNA
was incubated without ATP in NE at 30°C for 0 min to assemble H complexes or 20 and
40 min to chase H complexes into E complexes. Figure 3A shows MINX pre-mRNA
formed H complexes upon addition to NE (lane 1). Nearly all of the MINX RNA was
chased into E complexes following incubation for 20 and 40 min at 30°C (lanes 2 and 3).
In a time course experiment, anti-gal-l antiserum immunoprecipitated splicing competent
pre-mRNA assembled in H complexes at 0 time (Figure 3B, lane 5) and E complexes
after 20 through 60 min of incubation at 30°C (Figure 3B, lanes 6-8). Thus, complexes
formed in the absence of ATP with mobilities characteristic of H and E pro-splicing
complexes contain gal-1. Similar results for gal-3 were obtained (data not shown).

To confirm further that the immunoprecipitated complexes represented
spliceosomes, antisera specific for several splicing factors were used to probe the
precipitated fractions (Figure 4). Antiserum specific for gal-1 co-precipitated the Sm
B/B’ core polypeptides of snRNPs, hnRNP C1/C2 and a factor required for in the second

transesterification reaction Slu7. In contrast, neither Ran (a protein does not involved in

53

splicing, data not shown) nor gal-3 was detected in the immunoprecipitate. As expected,
gal-l antiserum precipitated gal-1. Similar results were obtained when the
immunoprecipitates of anti-gal-3 antiserum were analyzed by westem analysis (data not
shown).

Galectins should be detected in spliceosomes isolated by precipitation with
antiserum directed against another splicing factor. A reciprocal co-immunoprecipitation
experiment was performed using anti-Sm antisera (Figure 5). As expected, spliceosomes
selected by the Sm antiserum contained the splicing substrate, intermediates and products.
Immunoprecipitation with human IgG revealed only background levels of splicing RNAs.
Most importantly, gal-1 was detected in the Sm selected complexes, but not detected in
the material precipitated by the control human IgG. These data strongly support our

contention that galectins are splicing factors associated with the splicing machinery.

Galectin-1 and galectin-3 reside on distinct splicing complexes

Spliceosomes selected by galectin-specific antisera are a heterogeneous mixture
of complexes (e.g., spliceosomes containing pre-mRNA substrate would be expected to
contain different RNA and protein components than spliceosomes containing ligated
exon product). While the stoichiometry of most of the proteins in spliceosomes has not
been determined, it is assumed that under standard splicing conditions using a single
splicing substrate each distinct processing pathway intermediate will contain the same
complement of proteins. Thus, it is reasonable to predict that spliceosomes containing
gal-1 would also contain gal-3. However, in our previous depletion-reconstitution studies,

we showed that either gal-1 or gal-3 could restore splicing activity to a galectin-depleted

54

Figure 5: Immunoprecipitation of spliceosomes by anti-Sm antiserum.
Splicing reactions were incubated with ATP for 60 min and then were
immunoprecipitated with human anti-Sm antiserum or human IgG
(control). Bound fractions were eluted and RNA analyzed by gel
electrophoresis and autoradiography @anel A) and gal-1 detected by
western blotting (panel B).

55

Figure 5

Input
08m

11

L

hlgG

56

NE. Obviously, under these reconstitution conditions, spliceosomes containing a galectin
would contain only that particular member of the galectin family.

To distinguish between these two formal possibilities (spliceosomes precipitated
by one galectin antibody contain one or both nuclear galectins), we performed sequential
immunoprecipitations as outlined in Figure 6A. Standard splicing reactions were
incubated for 60 min and divided into two equal portions. One aliquot was immuno-
precipitated with anti-gal-l and the other with anti-gal-3. The unbound fractions were
then subjected to a second immunoprecipitation using the other galectin antiserum.
Radiolabeled RNA in the bound fractions from each immunoprecipitation was analyzed
(Figure 6B). Roughly the same quantity of spliceosomes was precipitated by the anti-gal-
1 antiserum in the two sequential selections (compare lanes 3 and 10). Similar results
were obtained following the two anti-gal-3 immunoprecipitations (compares lanes 4 and
8). In order to interpret these results, the efficiency of each galectin antiserum to
quantitatively immunoprecipitate its cognate antigen was determined. We analyzed the
bound and unbound fractions from the first immunoprecipitation for gal-1 and gal-3
(Figure 6C). The bound fraction from the first anti-gal-l precipitate showed only gal-1
(Figure 6C, lane 3) with no detectable gal-3. Further, the unbound fraction of this
precipitation showed nearly quantitative depletion of gal-1 (lane 6; the amount of gal-1 in
this fraction represents less than 10% of the total gal-1 in the reaction used for
immunoprecipitation). Similar results were obtained with gal-3. Analysis of the bound
fraction of the first anti-gal-3 precipitation showed only gal-3 (lane 4) and nearly all of
gal-3 was removed by this immunoprecipitation (lane 5; less than 15% of the total gal-3

in the reaction remained in the unbound fraction of the first precipitation). We interpret

57

Figure 6: Sequential immunoprecipitation of splicing complexes by gal-1 and
gal-3 antisera. Panel A: protocol for sequential immunoprecipitation. Splicing
reactions were incubated for 60 min with 32P-MINX and divided into equal
aliquots. The aliquots were incubated with the indicated antiserum as described
in Materials and Methods. Part of the unbound fraction was then incubated
with the other galectin antiserum. After washing, the bound material from the
first and second immunoprecipitations was eluted and analyzed for MINX
RNA (panel B). Lanes 1, 5, 7 and 9 represent aliquots of the bound fraction
used for immunoprecipitation (input). Lanes 2, 3, 4, represent the MINX RNA
bound to the indicated antiserum in the first immunoprecipitate and lanes 6, 8
and 10 represent MINX RNA bound to the indicated antiserum in the 2nd
immunoprecipitate. Panel C: Western blotting analysis of gal-l and gal-3 in
the bound (lanes 2, 3 and 4) and unbound (lanes 5, 6 and 7) fractions of the
first immunoprecipitation.

58

 

 

 

Figure 6

 

 

A B 1st IP 2nd IP
input Pl 061 063 input PI input «163 input 061
32P-MINX
+ w fi'
HeLa NE “

1s!
[p P' aGal-3 aGal-1

.uuh-

   

23,” p| aGaI-1aGal-3 ‘

 

 

 

 

12345678910

C 1st lP Bound 1st IP Unbound

NE Pl 061063 aG3 aG1 Pl NE

:W H Gal-3 i- -WW

w .. H rem -.
Gal-1 I l

mfllfllfluflll'lh
8

 

59

these data to indicate that gal-1 and gal—3 were quantitatively removed during the initial
immunoselection and that the two galectins reside on different splicing complexes.
Finally, spliceosomal RNAs could be immunoprecipitated by anti-Sm serum from the
material remaining after the two sequential galectin adsorptions (data not shown),
indicating that some spliceosomal complexes contained neither gal-1 nor gal-3.

As an additional evaluation of the nearly quantitative and specific removal of each
galectin by its respective antiserum, this experiment was repeated with the following
modification. The unbound fraction of the first immunoprecipitation was incubated with
the same antiserum as that used in the first antibody selection (i.e., unbound material of
anti-gal-l precipitation was rebound to anti-gal-l coated beads). Less than 5% of the
initially precipitated RNA was bound to the antiserum in the second round of
precipitation. This low level of binding to the same antiserum matched the low levels of
galectin found in the unbound material after the first precipitation.

It is important to note that these results provide another control for the specificity
of the spliceosomes precipitated by the anti-galectin antisera. The fact that each galectin
antiserum precipitates only its respective antigen indicates that these antibodies do not
precipitate nuclear proteins/complexes non-specifically and further suggests that galectins

do not bind to splicing complexes non-specifically.

Galectin-1 and galectin-3 do not bind MINX RNA directly
The findings described above could be explained by a direct interaction of the
galectins with the MINX pre-mRNA. If this interaction was with a unique site in the pre-

mRNA, then only one galectin would bind per pre-mRNA and spliceosomes containing

60

this pre-mRNA would have either gal-1 or gal-3 associated. To test for direct galectin-
MINX interactions, we used an electrophoretic mobility shift assay (Figure 7). 32P-MINX
RNA migrated in native polyacrylamide gels as shown in lane 1 of Figure 7A and B.
Incubation of MINX with recombinant human gal-1 (0.5 or 2.5 pg, Figure 7A, lanes 2
and 3, respectively) or recombinant gal-3 (0.8 or 4 pg, Figure 7B, lanes 2 and 3,
respectively) did not alter the mobility of MINX when compared to the mobility of the
substrate alone. Various incubation conditions (i.e., changing temperature, time of
incubation and incubation with or without ATP) yielded the same results. As a positive
control for altered mobility, MINX was incubated with NE for 15 min on ice. As
expected, MINX was assembled into an H complex that had a slower mobility in the
native gel (lane 4 of Figure 7A and B). We conclude that the association of galectins with
spliceosomes occurs through interaction with another splicing component rather than

through direct binding to the splicing substrate.

Amino terminal domain of gal-3 lblocks gal-1 association with spliceosomes

Splicing activity in a complete HeLa NE is inhibited in a dose dependent manner
by the addition of the ND of gal-3. At the highest concentration of ND, neither splicing
activity nor spliceosome complex formation could be detected even though gal-1 was
available (14). Our finding that each galectin resides on separate splicing complexes
suggests a mechanism for this dominant negative effect of the ND. Splicing requires
binding of galectins to another splicing factor. Likely this partner is shared by the
galectins in a mutually exclusive manner. We suggest that excess ND binds to this

partner and blocks the interaction of this partner with both nuclear galectins. To test this

61

Figure 7: Native gel analysis of splicing substrate incubated with
recombinant gal-1, recombinant gal-3 or NE. Panel A: 32P-MINX
was incubated on ice for 15 min with 0.5 (lane 2), 2.5 (lane 3) pg
recombinant human gal-1, HeLa NE ~25 pg protein (lane 4), or
nothing (lane 1). Panel B: 32P-MINX was incubated on ice for 15 min
with 0.8 (lane 2), 4.0 (lane 3) pg recombinant gal-3, HeLa NE ~25 pg
protein (lane 4) or nothing (lane 1). Following incubation, heparin
was added and the samples analyzed on 4% polyacrylamide native
gels followed by autoradiography. The migration of free MINX RNA
and MINX RNA in H-complexes (indicated between the two panels)
was detected by autoradiography.

62

Figure 7

A

- r61 r61 NE
0.5pg 2.5pg

‘ H

1

1234

Free MINX

63

- r63 r63 NE
0.8119 4119

“i“

. . ” Free MINX

prediction, we quantitated spliceosomes immunoprecipitated by anti-gal-l antiserum in
reactions inhibited by the ND of gal-3 (added as a GST fusion protein). The results are
shown in Figure 8. As previously reported (14), the GST-ND polypeptide inhibited
product formation nearly 100% (lane 5) compared to reactions incubated with (lane 3) or
without (lane 1) GST. The addition of GST to the reaction did not inhibit the precipitation
of spliceosomes by anti-gal-l (compare lane 4 to lane 2). In contrast, the addition of
GST-ND nearly completely inhibited the association of gal-1 with the splicing machinery
(compare lane 6 to lanes 2 and 4). Thus, the ND of gal-3 exerts its dominant negative

effect by regulating the incorporation of gal-1 (and gal-3) into splicing complexes.

Galectin association with spliceosomes is salt labile

The strength of the association of galectins with spliceosomes was evaluated in
relation to the stable association of the snRNPs with splicing complexes. Splicing
complexes formed after a 60 min splicing reaction (Figure 9, lane 1) were incubated with
each galectin antiserum or pre-immune serum in 60 mM KCl buffer (the buffer used for
optimal splicing efficiency). Aliquots of the antibody-bound spliceosomes were then
washed with 60 mM (lanes 2-5), 130 mM (lanes 6-9) or 250 mM (lanes 10-13) KCl
buffers. The bound fractions were eluted and analyzed for radiolabeled RNA. Salt
concentrations of 130 or 250 mM released most of the splicing substrate from the
galectin-selected columns (>90% of the spliceosomes were released as determined by
quantitation from phosphorimage analysis) whereas 130 mM KCl had no effect on
spliceosomes selected by anti-Sm antiserum. Even when the salt was increased to 250

mM KCl, nearly 20% of the snRNPs remained stably associated with spliceosomes. The

64

Figure 8: Analysis of spliceosomes immunoprecipitated by anti-gal-l
antiserum in reactions incubated with GST or GST-ND of gal-3.
Equivalent splicing reactions were incubated with no addition (lane 1,
input), GST (lane 3, +GST: input, 100 pM) or GST-ND (lane 5, +GST-
ND: input, 100 pM) for 60 min. Anti-gal-l beads were added to each
reaction and RNA in the bound fractions was analyzed by gel
electrophoresis and autoradiography (lanes 2, 4 and 6).

65

Figure 8

‘5
:-
III! E
e-I ._'. ""
5 “ .-
n- r:
.5 ’3 z
_ I. II I
I
‘5 a E; B B
E o ‘1? ‘4? S?
.- B
w nu...» w ~.........
.5...“
i“ r
“"l!‘.‘ ..
It ‘ qt. I'
mum.- u.-
‘H - limit
.223} 1 --------
l-tl."
W ‘ Maw

66

 

+GST- ND: aGal—l

Figure 9: Analysis of spliceosomes precipitated by various antisera at
increasing salt concentrations. HeLa NE was incubated with 32P-MINX for
60 min in the presence of ATP. Immobilized pre-immune serum (lanes 2,
6 and 10); anti-gal-l (lanes 3, 7 and 11); anti-gal-3 (lanes 4, 8 and 12) or
anti-Sm (lanes 5, 9 and 13) was incubated with the reactions in 60 mM
KCl-buffer for 60 min. Beads were then washed extensively with 60 mM
KCl-buffer (lanes 2-5); 130 mM KCl-buffer (lanes 6-9); or 250 mM KCl-
buffer (lanes 10-13). The bound fractions were eluted and RNA analyzed
by denaturing gel electrophoresis and quantitated by phosphorimage
analysis (Molecular Dynamics) following autoradiography. Lane 1
indicates the RNA subjected to immunoprecipitation.

67

Figure 9

60 mM KCI

130 mM KCI 250 mM KCI

 

input Pl 061 063 a

 

.1‘1

it

      

 

 

Sm Pl 061 063 aSm Pl 061 063 aSm

      

 

 

l , l

"smut

3‘15"

l" mw.
' ‘ wultth‘ ‘ “I"
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

68

BEEF

loss of spliceosomal RNAs from the antibody columns was due to dissociation of the
complexes from each galectin and not due to release of the galectins from their respective
antibody. At 130 mM KCI, virtually no gal-1 or gal-3 was released from the immobilized
antibodies compared to the galectins bound at 60 mM KCI. At 250 mM KCl, more than
70% of the galectins remained bound to the antibodies (data no shown). We conclude that
the association of galectins with the splicing machinery is sensitive to perturbation of
ionic strength.
Discussion

We previously have shown by depletion-reconstitution experiments that galectins-
] and -3 are required splicing factors (10, 11). Further, the splicing activity of the
galectins appears to be redundant in that either galectin can reconstitute splicing in a
galectin-depleted NE. Now we document a direct association of these galectins with
spliceosomal complexes. The key findings are (i) mono-specific galectin antisera
immunoprecipitate splicing substrate RNAs in H, E and active spliceosomes along with
Sm proteins of snRNPs, hnRNP C1/C2 and Slu7 (all known splicing factors), (ii)
spliceosomal complexes contain either gal-1, gal-3 or no galectin, (iii) neither galectin
interacts directly with the pre-mRNA substrate, (iv) the amino terminus of gal-3 exerts a
dominant negative effect on splicing by regulating the entry of gal-1 (and presumably
gal-3) into splicing complexes and (v) the association of galectins with spliceosomes is
salt sensitive compared to the stable association of the snRNPs.

The data presented are significant in evaluating the role of galectins as splicing
factors. First, these data support our hypothesis that nuclear galectins are indeed splicing

factors that can be incorporated into the canonical model for pre-mRNA splicing (1). The

69

fact that both galectin antibodies co-immunoprecipitate pre-mRNA associated with early
(i.e., H and E) complexes illustrates the initial entry of galectins during spliceosome
assembly. While a precise molecular function of galectins during spliceosome assembly
is not known, it appears to be in the recruitment or supplying of snRNPs to pre-mRNA
based on two observations: (i) depletion of galectins from splicing extracts inhibits
transition of early (e.g., H and/or E) complexes to active spliceosomes (1), and (ii)
galectins are associated with gemin4 in SMN complexes (14) which are implicated in
recycling snRNPs to pre-mRNA in the early commitment complex (19).

Since the anti-galectin antibodies precipitated not only the pro-mRNA substrate
but also the intermediates and products of the splicing reaction generated on the
spliceosome, our results suggest that gal-1 and gal-3 are associated with the assembled
machinery throughout the reaction cycle. It is clear that these galectin-containing
complexes are spliceosomes as the hnRNP C1/C2 polypeptides (known splicing factors
[5-9, 20]), the snRNP-specific Sm proteins and Slu7 (2, 5-9) co-precipitate with the
substrate intermediates and products. Results of three controls strengthen our
interpretation of the data. First, the nuclear shuttling protein Ran, which has not been
found associated with spliceosomes, is not co-precipitated by the galectin antisera.
Second, gal-l-containing spliceosomes are not precipitated by antibodies specific for gal-
3 and vice versa. These results indicate (i) the galectin antisera are mono-specific and (ii)
galectins do not adhere non-specifically to splicing complexes. Third, spliceosomes
isolated by precipitation with antibodies directed against the Sm polypeptides of snRNPs

co-precipitate galectins.

70

An obvious question of our contention that galectins are splicing factors is that
they have not been identified in any of the proteomic analyses of spliceosomes (5-9).
Several reasons can be offered to explain this discrepancy. First, most of the spliceosome
isolation procedures use 150 — 250 mM salt during binding or washing to select stable
complexes. The rationale has been to correctly identify components with a stable
association in the spliceosome, rather than catalogue all associations. As we have shown
(Figure 9), ionic conditions greater than 60 mM release the galectins from spliceosomal
complexes. We contend the buffer conditions we use for immunoprecipitation (which are
optimal for in vitro splicing activity) allow a more complete cataloguing of spliceosomal
proteins. Could the transient/loose association of galectins hint to a regulatory role?
Second, the galectins may be in low abundance in spliceosomes. Our observation that not
all spliceosomes contain galectins speaks to this point. Third, it is possible that galectins
only assist in initiating spliceosome assembly (i.e., only associate with a complex
containing the pro-mRNA substrate) and are not stable components of active splicing
complexes. Only a thorough and careful evaluation of these early complexes would
reveal this association. Finally, the stringency set for the identification of peptides and
subsequent database searches results in missing members of a complex. For example, of
the four massive proteomic analyses published, none have detected the U6-associated
polypeptide LSm5 and there are several instances where one of the four studies detected a
core spliceosomal component and the other three did not.

Other significant aspects of our findings include providing an explanation for
functional redundancy of the galectins and hinting at the nature of the spliceosome-

associated binding partner for the galectins. Reconstitution of a galectin-depleted NE can

71

be achieved by either gal-1 or gal-3 (10, 11). The sequential immunoprecipitation data
provide experimental proof for the exclusive incorporation of only a single galectin into a
splicing complex in a complete (i.e., non-depleted) splicing extract. Thus, functional
redundancy means spliceosomes contain only one galectin. We show that the ND of gal-3
regulates the entry of gal-1 into spliceosomes. In aggregate, these data suggest the
galectins share a common binding partner. This partner is probably a polypeptide splicing
factor that weakly interacts with the galectins. In a splicing extract, gal-3 interacts with
this partner via its two domains (the ND which contains the PGAYPGXXX repeats of
unknown function and the carboxyl terminus which is the CRD) whereas gal-1 only binds
via its single CRD. The observation that gal-3 is 8 — 10 times more efficient in
reconstituting splicing activity in galectin-depleted extracts compared to gal-1 supports
this contention (11). Addition of excess ND binds to this common partner and blocks
binding of gal-1 or gal-3. Abrogation of gal-1 and gal-3 binding results in inhibition of
splicing. It remains to be determined whether the galectin—binding partner is assembled
into splicing complexes when bound to the isolated ND.

Crucial to providing a mechanistic interpretation of these data regarding the
association of galectins with spliceosomal complexes is the identification of the splicing
partner for the galectins. While we have identified gemin4 as a galectin binding protein
(14), neither gemin4 nor other members of the SMN complex (19) have been identified
as spliceosomal components in proteomic analyses (5-9). Is this due to the fact that
gemin4 and interacting proteins are not spliceosomal proteins or, as with the galectins,
that they are weakly associated with spliceosome complexes and released under the

conditions used for spliceosome isolation? From a different perspective, gal-1 has been

72

shown to be a component of the nuclear matrix (21) that has been proposed to serve as a
scaffold for the splicing process. The nuclear matrix partner with which gal-1 interacts is
unknown. Also, proteomic analysis of interchromatin granule clusters (IGC) has
identified a galectin as a member of this nuclear organelle. Over 80% of the proteins
identified as IGC components play a role in RNA biogenesis with over 50% having a
splicing function (22). Both of these findings lend additional, albeit indirect, support to
our assertion that the galectins are splicing factors.

Galectin-containing spliceosomes have been precipitated with several different
galectin-specific antisera using three conditions to assemble various splicing complexes.
The association of galectins with spliceosomes is through a salt-sensitive protein-protein
interaction rather than a galectin-splicing substrate interaction. The commonly shared
binding partner/ splicing factor explains the mutually exclusive incorporation of gal-1 or
gal-3 into splicing complexes and the dominant negative inhibition of splicing

demonstrated by the ND of gal-3.

Acknowledgments
Jung Park contribute more than one third of the work in this paper. We also thank Kevin
Haudek, Rich Gray and Patty Voss for their invaluable input and comments on this
manuscript. The work has been supported by grants MCB-0092919 (RJP) from the
National Science Foundation and GM-38740 (JLW) from the National Institutes of

Health.

73

References

#

. Brow, D. A. (2002) Allosteric cascade of spliceosome activation. Ann. Rev. Genet.,
36, 333-360.

2. Chua, K., and Reed, R. (1999) Human step 11 splicing factor hSlu7 functions in
restructuring the spliceosome between the catalytic steps of splicing. Genes Dev.,
13, 841-850.

DJ

. Konarska, M. M., and Query, C. C. (2005) Insights into the mechanisms of splicing:
more lessons from the ribosome. Genes Dev., 19,2255-2260.

A

. Hastings, M. L., and Krainer, A. R. (2001) Pre-mRNA splicing in the new
millennium. Curr. Opin. Cell Biol., 13, 302-309.

5. Neubauer, G., King, A., Rappsilber, J ., Calvio, C., Watson, M., Ajuh, P., Sleeman, J .,
Lamond, A., and Mann, M. (1998) Mass spectrometry and EST-database

searching allows characterization of the multi-protein spliceosome complex.
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6. Hartmuth, K., Urlaub, H., Vomlocher, H-P., Gentzel, M., Wilm, M., and Luhrmann, R.
(2002) Protein composition of human prespliceosomes isolated by a tobramycin
affinity-selection method. Proc. Natl. Acad. Sci. USA, 99, 16719-16724.

7. Rappsilber, J ., Ryder,U., Lamond, A., and Mann, M. (2002) Large-scale proteomic

analysis of the human spliceosome. Genome Res., 12, 1-15.

8. Zhou, Z., Licklider, L. J ., Gygi, S. P. and Reed, R (2002) Comprehensive proteomic
analysis of the human spliceosome. Nature, 419, 182-184.

9. Makarov, E. M., Makarova, O. V., Urlaub, H., Gentzel, M., Will, C. L., Mann, M.,
and Luhrmann, R. (2002) Small nuclear ribonucleoprotein remodeling during
catalytic activation of the spliceosome. Science, 298, 2205-2208.

10. Dagher, S.F., Wang, J. L., and Patterson, R. J. (1995) Identification of galectin-3 as a
factor in pre-mRNA splicing. Proc. Natl. Acad. Sci. USA, 92, 1213-1217.

11. Vyakamam, A., Dagher, S. F ., Wang, J. L., and Patterson, R. J. (1997) Evidence of a
role for galectin-l in pre-mRNA splicing. Mol. Cell. Biol., 17, 4730-4737.

12. Barondes, S.H., Castronovo, V., Cooper, D. N. W., Cummings, R. D., Drickamer, K.,
Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes, C., Kasai, K., Leffler, H., Liu, F.-
T., Ictan, R., Mercuric, A. M. , Monsigny, M., Pillai, S., Poirier, F., Raz, A.,
Rigby, P. W., Rini, J. M., and Wang, J. L. (1994) Galectins: A family of animal [3-
galactoside-binding lectins. Cell, 76, 597-598.

74

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Vyakamam, A., Lenneman, A. J ., Lakkides, K. M., Patterson, R. J ., and Wang, J. L.
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Park, J. W., Voss, P. G., Grabski, S., Wang, J. L., and Patterson, R. J. (2001)
Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the
SMN protein. Nucleic Acids Res., 29, 3595-3602.

Hirabayashi, J., Ayaki, H., Soma, G., and Kasai, K. (1989) Production and
purification of a recombinant human 14 kDa beta-galactoside-binding lectin.
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Agrwal N, Sun Q, Wang SY, Wang JL. (1993) Carbohydrate-binding protein 35. 1.
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Zillman, M., Zapp, M. L., and Berget, S. M. (1988) Gel electrophoretic isolation of
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Das, R., Zhou, Z., and Reed, R. (2000) Functional association of U2 snRNP with the
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Pellizzoni, L., Kataoka, N., Charroux, B., and Dreyfitss, G. (1998) A novel function
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Choi, Y.D., Grabowski, P. J ., Sharp, P. A., and Dreyfuss, G. (1986) Heterogeneous
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75

Chapter 3

Dissociation of the splicing and the carbohydrate-binding
activities of galectin-1

ABSTRACT

A site-directed mutant of galectin-1 (Gall), in which an asparagine at residue 46
is replaced by aspartic acid, was expressed and purified as a fusion protein with
glutathione S-transferase (GST). This mutant, designated as GST-Gall(N46D), was
compared with the corresponding fusion protein containing wild-type galectin-1, GST-
Ga11(WT), in three assays: (a) binding to asialofetuin-Sepharose, as a test of the
carbohydrate-binding activity; (b) reconstitution of splicing in a galectin-depleted nuclear
extract of HeLa cells, as a test of the in vitro splicing activity; and (c) reconstitution of
splicing in a nuclear extract of HeLa cells containing 150 mM thiodigalactoside, as a test
of sensitivity to saccharide inhibition. The binding of GST-Gall(N46D) to asialofetuin-
Sepharose was less than 10% of that observed for GST-Gall(WT), indicating that the
mutant was deficient in carbohydrate-binding activity. Both GST-Gall(WT) and GST-
Ga11(N46D) were equally efficient, however, in reconstitution of splicing activity in a
galectin-depleted nuclear extract, suggesting that carbohydrate-binding, per se, was not
required for the splicing activity. Previous experiments had shown that the endogenous
splicing activity of HeLa cell nuclear extracts can be inhibited by the addition of
thiodigalactoside. GST-Ga11(WT) cannot overcome this inhibition. In contrast, the

addition of GST-Gall (N46D) to a thiodigalactoside-inhibited nuclear extract resulted in

76

splicing of the pre-mRNA substrate. Additional experiments show that Gall is
incorporated into H, E and active splicing complexes. Addition of thiodigalactoside
released Gall from H splicing complexes, but not from committed complexes (i.e., E and
active spliceosomes). These results indicate that the carbohydrate-binding and the

splicing activities of galectin-1 can be dissociated.

INTRODUCTION

Galectins are a family of widely distributed proteins that (a) bind to B-
galactosides and (b) contain characteristic amino acid sequences in the carbohydrate
recognition domain of the polypeptide (1, 2). In previous studies, we had reported the
localization of galectin-1 (Gall)l and galectin-3 (G313) in the cell nucleus (3, 4) and
identified them as two of the many proteins required for the splicing of pre-mRNA,
assayed in a cell-free system (5, 6). The key findings from these studies included: (a)
nuclear extracts (NE) derived from HeLa cells, capable of carrying out splicing of pre-
mRNA, contained both Gall and G313; (b) depletion of both galectins from NE, either by
lactose (Lac) affinity chromatography or by antibody adsorption, resulted in the
concomitant loss of splicing activity; and (c) either recombinant Gall or recombinant
Gal3 was able to reconstitute splicing activity in a galectin-depleted extract.

More recently, we have identified Gall and Gal3 as novel components of
macromolecular complexes containing the survival of motor neuron (SMN) protein,
Gemin2 and the core snRNP polypeptides bearing the Sm epitope (7). Both galectins

interact directly with at least one component of these complexes, Gemin4. The SMN-

77

containing complexes are found in the cytoplasm and in discrete nuclear bodies called
gems (gemini of coiled bodies) (8-10). In the former, the SMN complex plays a critical
role in the biogenesis of snRNPs (11, 12). In the nucleus, SMN is required for pro-mRNA
splicing by supplying snRNPs to the H-complex in the assembly of spliceosomes (13, 14).
This H-complex juncture is indeed where galectins appear to be required, as
demonstrated by the effect of galectin depletion (6) and by the effect of addition of the
NHz-terminal domain of galectin-3, which exerts a dominant negative effect on
spliceosome assembly and the splicing reaction (7).

In the course of our studies, we had observed and reported that saccharides which
bind to Gall and Gal3 with high affinity, such as Lac and thiodigalactoside (TDG),
inhibited the cell-free splicing reaction when added to a complete NE (5, 6). In contrast,
saccharides that do not bind to Gall and Gal3, such as cellobiose, failed to have any
effect. Does inhibition of splicing by Lac and TDG indicate that the nuclear ligand for the
galectins interacts with the carbohydrate-binding site and that it is competitively
displaced upon Lac/TDG addition? Or, does Lac/TDG addition alter the conformation of
the galectins, causing the release or altered interaction with a nuclear ligand bound via
protein-protein, rather than protein-carbohydrate, interactions? In the present
communication, we report the identification of a site-directed mutant of Gall, deficient in
carbohydrate-binding activity, that is still capable of functioning in the splicing assay.
We show that TDG can release Gall from H splicing complexes, but not from committed
complexes. The results suggest that carbohydrate-binding, per se, is not required for the

splicing activity of galectins.

78

EXPERIMENTAL PROCEDURES

Glutathione S-transferase (GST) Fusion Proteins

The cDNAs for wild-type (WT) and Asn46Asp (N46D) mutant forms of human
Gall have been described (15). Each cDNA was subcloned into the BamHI restriction
site of the pGEX-ZT vector (Pharmacia) to produce a fusion protein between GST and
Gall: GST-Gall(WT) and GST-Gall(N46D). Each of the constructs was subjected to
DNA sequencing to verify: (a) the juncture of the fusion protein between GST and Gall;
and (b) the wild-type and mutant amino acid at residue 46.

The constructs were transformed into E. coli (strain BL21-codon Plus (DE3);
Stratagene) and GST-Ga11(WT) and GST-Ga11(N46D) were purified from 1 liter cultures
on the basis of GST binding to glutathione-agarose beads (Pierce). The purity of the
protein preparations was assessed by SDS-PAGE (16). The polypeptides were revealed
by silver staining (17) or by immunoblotting (7), following procedures that have been
described previously. Affinity purified polyclonal rabbit anti-Gall and anti-GST
antibodies were used for the immunoblotting.

To generate the antibodies, the immunogen used was GST-Gall(WT) purified on
the basis of binding to two columns: (a) glutathione-agarose and elution with glutathione;
and (b) Lac-agarose (Sigma) and elution with Lac. Approximately 70 ml of antisera,
pooled from four bleeds of rabbit #55, were subjected to ammonium sulfate fractionation
(50% of saturation). The immunoglobulin-containing precipitated fraction was
solubilized in phosphate-buffered saline (PBS) and passed over a 5-ml column of GST-

agarose. The unbound (flow-through) fraction was immediately loaded over the same

79

column (six passes over the same column to insure binding). The bound fraction was
eluted with 0.1 M glycine-HCl (pH 2.2) and this was dialyzed immediately against PBS
to neutralize the pH. The bound and eluted material from the GST affinity column is
designated as affinity purified anti-GST. Purified GST-Gall(WT) (the immunogen) was
bound to glutathione-agarose and covalently cross-linked with dimethylpimelimidate (20
mM; Pierce). The reaction was carried out in 0.2 M sodium borate (pH 9) for 1 hour at
room temperature; the cross-linked beads were washed twice with 0.2 M ethanolamine,
pH 8, followed by a 2-hour incubation at room temperature in the same buffer to block
unreacted groups. The unbound fractions of the antisera, depleted of anti-GST antibodies,
were passed over this GST-Gall(WT) affinity column (six passes to insure binding). The
bound and eluted material from this column is designated as affinity purified anti-Gall.
At 122000, 125000, and 1:10,000 dilutions, this affinity purified anti-Gall blots Gall in

NE of HeLa cells and purified GST-Gall(WT) but does not blot GST.

Assay of Carbohydrate-binding Activity

The preparation of the saccharide affinity beads, asialofetuin (ASF)-Sepharose 43,
has been described (18). Approximately 180 nmoles of ASF were coupled per ml of
Sepharose beads. GST proteins (350 ng each of GST, GST-Ga11(WT), and GST-
Gall(N46D)) were incubated with 35 pl of ASP-Sepharose for 2 hours at 4 °C. The
incubations were canied out in 60% buffer D (buffer D is 20 mM Hepes, pH 7.9, 20%
glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenyl-methylsufonyl fluoride, and 0.5
mM dithiothreitol) containing 0.1% NP-40 (Pierce) in the presence and absence of Lac
(100 mM). The material not bound to the beads was removed by centrifugation (1000 x g)

and resuspension; the beads were washed four times in 60% buffer D containing 0.1%

80

NP-40. The GST proteins bound to the beads were subjected to SDS-PAGE and
immunoblotting with anti-GST and anti-Gall antibodies.

The chemiluminescent signal provided by horseradish peroxidase conjugated to
the secondary antibody was detected using the Western Lightning reagent (Perkin Elmer
Life Sciences). This signal was quantitated with a BioRad model GS505 Molecular
imager system and associated software. Known amounts of GST, GST-Ga11(WT), and
GST-Ga11(N46D) were used to establish standard curves. The quantitative value of the
immunoblotted band derived from the incubation carried out in the presence of Lao
represented non-specific binding not inhibitable by Lac; this accounted for about 3% of
the total binding for both the wild-type and mutant proteins. Lac-inhibitable specific
binding was calculated by subtracting this value from the total binding.

Assay of Splicing Activity

HeLa S3 cells were grown in suspension culture by the National Cell Culture
Center (Minneapolis, MN). NE was prepared in buffer C (20 mM Hepes, pH. 7.9, 25%
glycerol, 0.42 M NaCl, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM phenylmethylsufonyl
fluoride, 0.5 mM dithiothreitol), as described by Dignarn et al. (19). Protein
concentrations were determined by the Bradford assay (20); in this study, the protein
concentration of the NE was ~4 mg/ml.

NEs were depleted of galectins by adsorption on ASE-Sepharose (18). The beads
(150 pl) were washed with 20 mM Hepes, pH 7.9, 0.5 M NaCl; 30 pl NE were added and
incubated on ice for 20 minutes in disposable spin columns (Millipore). The unbound
fraction was removed and the beads were washed with 12 p1 of 60% buffer D adjusted to

0.42 M NaCl and this wash was combined with the unbound fraction. Aliquots of

81

nondepleted NE and unbound fractions of the depletion were dialyzed in a microdialyzer
against 60% buffer D for 40 minutes at 4 °C using a dialysis membrane with a 10-kDa
cutoff. In reconstitution experiments, GST, GST-Gall(WT), or GST-Gall(N46D) were
added to the unbound fractions prior to dialysis. The dialyzed fractions were then assayed
for splicing activity.

The plasmid used to transcribe the MINX pre-mRNA substrate was obtained from
Dr. Susan Berget (Baylor College of Medicine, Houston, TX) (21). The MINX pre-
mRNA was labeled with [32P]GTP and the monomethyl cap was added during SP6
polymerase (Gibco BRL) transcription. Splicing reaction mixtures contained a total
volume of 10 pl: dialyzed NE (4 pl) or unbound fraction (8 pl), [32P]MINX pro-mRNA,
2.5 mM MgC12, 1 mM ATP, 20 mM creatine phosphate, 0.5 mM dithiothreitol, and 20 U
of RNasin (Promega). In experiments testing the effect of exogenously added
carbohydrates, the extracts were incubated with 150 mM TDG or 150 mM cellobiose for
5 minutes at room temperature prior to the addition of pre-mRNA substrate. Splicing
reactions were incubated at 30 °C for 45 minutes. Proteinase K-SDS solution was added
to a final concentration of 4 mg/ml and 0.1%, respectively. The sample was incubated at
37 °C for 15 minutes. Each sample was then diluted to 100 pl with 125 mM Tris, pH 8, 1
mM EDTA, 0.3 M sodium acetate. RNA was extracted with 200 pl of phenol-chlorofonn
(50:50 (vol/vol)), followed by 200 pl chloroform. RNAs were precipitated with 400 pl of
ethanol at -80 °C. The extracted RNAs were subjected to electrophoresis through 13%
polyacrylamide (bisacrylamide-acrylamide, 1.9:50 (wt/wt)), 8.3 M urea gels, followed by

autoradiography.

82

Preparation of H complexes involved addition of pre-mRNA to NE in buffer D in
the absence of ATP, creatine phosphate and Mng and incubation on ice for 45 min. E
complexes were formed by incubation at 30 °C of a reaction identical to that used for H
complex formation. Active spliceosomes were formed as above.

Quantitation of the amount of radioactivity was carried out on a STORM
phosphorimager (Molecular Dynamics). The percent product formation was calculated by
dividing the radioactivity present in the final product (ligated exon l-exon 2) by the total
radioactivity present in the pre-mRNA substrate, the splicing intermediates (exon 2-lariat
and exon 1), and the product.

Immunoprecipitation of Splicing Complexes

Polyclonal rabbit antiserum to Gall or pre-immune serum were attached to
protein G-agarose beads (described in chapter 2). After extensive washing, the beads
were incubated with preformed H, E or active spliceosomes in the presence or absence of
TDG or cellobiose. After washing, the bound material was eluted with PAGE-SDS

sample buffer and aliquots assayed for proteins or splicing substrate RNAs.
RESULTS

GST-Gall(N46D) is Deficient in Carbohydrate-binding Activity
Hirabayashi and Kasai (15) had demonstrated that substitutions at highly

conserved residues of the carbohydrate recognition domain of human Gall, such as
Asn46, Glu71, and Arg73, resulted in loss of saccharide-binding activity. The cDNAs

corresponding to the WT and N46D mutant were each subcloned into the pGEX-2T

83

vector to produce a fusion protein between GST and Gall so that we can take advantage
of the easy purification of the proteins on the basis of GST binding to glutathione beads.
Nucleotide sequencing analysis of the constructs verified the sequence of the fusion
protein; in particular, residue 46 in the Gall sequence was confirmed to be Asn and Asp,
respectively for GST-Gall(WT) and GST-Gall (N46D). In parallel, we also expressed
GST from the same vector to be carried as a control in the functional tests below.

On SDS-PAGE and silver staining (Figure 1, panel A, lanes 1-3), each of the
purified GST proteins yielded a single band with a mobility corresponding to the
expected molecular weight: GST, ~27 kDa; GST-Ga11(WT), ~42 kDa; and GST-
Gall(N46D), ~42 kDa. Irnmunoblotting with affinity purified anti-Gall antibodies
yielded a single band at the same molecular weight for GST-Gall(WT) and GST-
Gall(N46D) (Figure 1, panel B, lanes 2-3). No reaction was observed between anti-Gall
and GST (Figure 1, panel B, lane 1). Finally, immunoblotting of the respective GST
protein preparations with affinity purified anti-GST antibodies yielded the same single
band patterns as were observed by silver staining (Figure 1, panel C, lanes 1-3). These
results establish the purity of the protein reagents to be compared in the functional assays
below.

Purified GST-Gall(WT) and GST-Gall(N46D) were compared in terms of their
binding to ASF-Sepharose beads. After washing, the material bound to the beads was
subjected to SDS-PAGE and immunoblotting with anti-GST antibodies. Using known
amounts of GST-Gall (WT or mutant) to establish standard curves, we quantitated the

Lac-inhibitable binding, as well as binding not inhibitable by Lac. The latter accounted

84

Figure 1: Characterization of the preparations of fusion proteins
containing wild-type galectin-1 or its N46D mutant by SDS-PAGE.
Lane 1: GST; lane 2: GST-Ga11(WT); and lane 3: GST-Gall(N46D).
The proteins (30 ng in each lane) were electrophoresed through 12.5%
acrylamide gels. Panel A: silver staining; Panel B: immunoblotting
with affinity-purified anti-Gall antibodies; Panel C: immunoblotting
with affinity-purified anti-GST antibodies. The binding of the primary
antibodies in panels B and C were revealed with horseradish
peroxidase—conjugated goat anti-rabbit immunoglobulin and the
enhanced chemiluminescent system. The positions of migration of
molecular weight standards (80 kDa; 52 kDa; 35 kDa; 30 kDa; and 22
kDa) are indicated on the left.

85

Figure 1

 

86

for about 3% of the total binding observed for both the WT and mutant proteins. In terms
of Lac-inhibitable binding, 10-15% of the GST—Gall(WT) added to the assay was bound
specifically; in contrast, less than 1% of the GST-Gall(N46D) added to the assay was
bound in a Lac-inhibitable fashion. More importantly, we found that the binding of the
N46D mutant was drastically reduced (to less than 10%) relative to the wild-type protein
(Figure 2). The level of binding of GST-Gall(N46D) was similar to that observed for
GST. The same overall conclusion was obtained using either anti-GST antibodies (Figure
2) or anti-Gall antibodies for the quantitation (the latter obviously could not detect GST
itself, as was shown in Figure 1, panel B, lane 1).

Reconstitution of Splicing in Galectin-depleted NE by GST-Gall(WT) and GST-
Gall(N46D)

NE was prepared in buffer C, which contained 0.42 M NaCl to dissociate splicing
or RNP complexes. This NE was incubated with ASP-Sepharose. Western blotting
analysis with antibodies against Gall and Ga13 documented that both proteins were
present in the NE and in the bound fraction of the affinity beads. Neither Gall nor Ga13
was detected in the unbound fraction. Thus, the galectins were quantitatively depleted
from the extract.

NE, depleted of the galectins by saccharide affinity adsorption, was also depleted
in terms of splicing activity (Figure 3, lanes 1-2). We had previously documented that
GST, by itself, had no effect on the cell—free splicing assay (7) so the effects of purified
GST-Gall(WT) and GST-Gall(N46D) could be directly compared. The same
preparations of the WT and mutant proteins that exhibited drastic differences in

saccharide-binding activity (Figure 2) both reconstituted splicing in the galectin-depleted

87

Figure 2: Comparison of the carbohyrate-binding activity of GST-
Gall(WT) and GST-Gall(N46D). GST proteins (350 ng each of GST,
GST-Gall(WT) and GST-Gall(N46D)) were incubated with ASF-
Sepharose for 2 hours at 4 °C in 60% buffer D containing 0.1% NP-40.
Parallel incubations were carried out in the presence and absence of
100 mM Lac. The GST proteins bound to the ASF-beads were
quantitated by Western blotting with anti-GST. The values shown
represent Lac-inhibitable specific binding.

88

Figure 2

50

 

40-

ng bound

10-

 

GST

 

GSTGi
(W0

89

 

GST61’
(N460)

 

Figure 3: Comparison of the splicing activities of NE, NE after
adsorption on ASF-Sepharose, and depleted NE reconstituted with
GST, GST-Gall(WT) and GST-Gall(N46D). Lane 1: the complete
(non-depleted) NE; lanes 2-5: the unbound (UB) fractions from the
ASE-Sepharose adsorption, assayed without (lane 2) and with
reconstitution with GST (lane 3), GST-Gall(WT) (lane 4) and GST-
Gall(N46D) (lane 5). The concentration of the GST proteins was 20
pM. Products of the splicing reactions were analyzed by
electrophoresis through a 13% polyacrylamide-urea gel and
autoradiography. The positions of migration of the pre-mRNA
substrate, the splicing intermediates (exon 1 and lariat-exon 2), and
mature RNA product are indicated on the right.

90

Figure 3

0
i
l 1
1|i' ill 1.1

UB of ASF

l,

.H‘L, ‘

r l- ‘
"illllll‘ -‘=
‘ ti 1 r

l

 

91

ll

l

l

NE (Figure 3, lanes 4-5). In contrast, GST by itself could not reconstitute the splicing
activity in the depleted NE (Figure 3, lane 3). The concentration of GST proteins used in
the reconstitution was 20 pM. At this concentration, the level of product formation (~7%)
in the reconstitution assay was comparable to that achieved previously with recombinant
galectin-1 (5).
Effect of GST-Gall(N46D) on NE Whose Splicing Activity is Inhibited by TDG

The above results suggest that the N46D mutant of Gall, deficient in
carbohydrate-binding activity, was still functional in terms of its splicing activity. A
confirmation of this notion was obtained when we carried out the splicing assay in NE
containing TDG (150 mM). As we had shown previously (5, 6), TDG inhibited the
splicing activity of a complete (non-depleted) NE when added to the cell-free assay
whereas cellobiose, which does not bind to the galectins, failed to yield the same effect.
The level of product formation was much higher in NE containing cellobiose (~30%;
Figure 4, lane 2) than the corresponding extract containing TDG (~1%; Figure 4, lane 1).

Addition of GST-Gall(WT), which can bind to Oligosaccharide-bearing ligands
(Figure 2), did not overcome the inhibitory effect of TDG on splicing (Figure 4, lanes 3-
5). In contrast, GST-Gall(N46D), which was deficient in carbohydrate binding (Figure 2),
was not sensitive to inhibition by the saccharide. Addition of the mutant fission protein
resulted in a dose-dependent increase in splicing activity (Figure 4, lanes 6-8).
Effect of TDG on the Association of Call with Splicing Complexes

If the carbohydrate binding function is not required for the splicing function, how
do galectin-specific saccharides inhibit the splicing activity in a NE? Since there are no

known nuclear glycoconjugates for galectins to bind in their splicing role, we believe that

92

Figure 4: Comparison of the effects of cellobiose, TDG, GST-
Ga11(WT) and GST-Gall(N46D) on the splicing activity of NE.
Lane 1: TDG addition (150 mM); lane 2: cellobiose addition (150
mM); lanes 3-5: TDG (150 mM) and GST-Gall(WT) (10, 20, and
30 pM); lanes 6-8: TDG (150 mM) and GST-Gall (N46D) (10, 20,
and 30 pM). Products of the splicing reactions were analyzed by
electrophoresis through a 13% polyacrylamide-urea gel and
autoradiography. The positions of migration of the pre-mRNA
substrate, the splicing intermediates, and mature RNA product are
indicated on the right.

93

Figure 4

GST- GST-
Gl(WT) Gl(N46D)
10 20 30 10 20 30pM

TDG + - + + + + + +
Cellobiose +

 

94

they participate by interacting with a splicing partner through protein-protein interactions.
Addition of saccharides with affinity for galectins would directly or indirectly interfere
with this required interaction.

One way to test this suggestion is to determine whether galectin-specific
saccharides release galectins that are associated with splicing complexes. As shown
previously, galectins are assembled in splicing complexes throughout the splicing
pathway (see chapter 2). Three distinguishable splicing complex intermediates can be
assembled using NE: these are the H, E and active complexes. H complexes form
immediately upon addition of pre-mRNA to a NE in the absence of ATP or elevated
temperatures. The H complexes are chased into E (early) complexes following incubation
at 30 °C. The E complexes are functionally committed and the addition of ATP converts
them to active spliceosomes, another heterogeneous collection of complexes that
catalyzes splicing chemistry.

H, E and active complexes were formed and Gall incorporation monitored by
immunoprecipitation with polyclonal anti-Gall antiserum. The results are shown in
Figure 5. Gall is associated with each complex (lanes 3, 7 and 11). We now determined
whether a saccharide with high affinity for galectins (TDG) could dissociate Gall from
these splicing complexes. As a negative control, the effect of a disaccharide with no
detectible galectin binding activity (cellobiose) was compared. Cellobiose had no effect
on Gall assembled into any of the splicing complexes (lanes 5, 9 and 13). In contrast,
TDG can release Gall from H complexes (compare lane 8 to lane 7). Interestingly, TDG

had no effect on Gall in E (compare lane 12 to lane 11) or active complexes (compare

95

lane 4 to 5). Once Gall is incorporated into complexes committed to the splicing pathway,

it is inaccessible for interaction with specific saccharide ligands.

96

Figure 5: Comparison of the effects of cellobiose and TDG on the
release of Gall from splicing complexes. Lanes 1, 6 and 10: NE
used as input material for each class of splicing complex; lanes 2-5,
active spliceosomes; lanes 6-9, H complexes; lanes 10-13, E
complexes. Lane 2 was precipitated with pre-immune serum; lanes
3-5, 7-9, and 11-13 were precipitated with anti—Gall in the presence
of cellobiose (150 mM) (lanes 5, 8 and 13); TDG (150 mM) (lanes
4, 7 and 12); or no additions (lanes 3, 7 and 11).

97

Figure 5

1

 

3N

Active H E
3 3
o -l 8 z o 3 g z 3 _. 8
E 3 U = m 3 = m a U =
0 Q o o o 0 ¢ 0 o

wwwmw

23455789101112“

98

DISCUSSION

The key findings of the present study include: (a) the N46D mutant of Gall,
expressed as a fusion protein with GST, is deficient in carbohydrate-binding activity,
compared to the WT protein; (b) GST-Gall(N46) can reconstitute splicing activity in NE
depleted of galectins, just like GST-Gall(WT); (c) unlike the WT protein, GST-
Gall(N46D) is insensitive to the presence of TDG and therefore, can overcome the
inhibition of splicing in NE containing the saccharide; and (d) Gall can be released from
H splicing complexes by TDG but it cannot be released from committed splicing
complexes (E or active spliceosomes). These finding represent the first demonstration of
a function for a galectin that is independent of carbohydrate-binding activity.

In previous studies, Hirabayashi and Kasai carried out site-directed mutagenesis
on the human Gall cDNA to generate the N46 mutant (15). When bacterial lysates
expressing this mutant were passed over asialofetuin-Toyopearl columns, Gall(N46D),
as detected by Western blotting, was found exclusively in the flow-through fraction
together with the bulk of the E. coli proteins. There was no sign of retardation on the
affinity columns that might reflect some retention of weak carbohydrate-binding capacity.
Thus, it was concluded that substitution of Asn by Asp at residue 46 resulted in complete
loss of saccharide-binding activity (15, 22). Like the parent mutant protein, Gall(N46D),
our present fusion construct GST-Gall(N46D) also resulted in a polypeptide deficient in
binding to asialofetuin. The level of binding ofGST-Ga11(N46D) was found to be similar
to that observed for GST. That residue 46 of Gall is critical in saccharide-binding is

consistent with the results of X-ray crystallographic analysis of the carbohydrate

99

recognition domain of the galectins, in which the Asn residue at this position serves as an
acceptor of a hydrogen bond from the hydroxy group at C-4 of galactose (23 - 26).

In any site-directed mutagenesis experiment, a critical issue is whether the
mutation has caused a disruption of the overall folding of the polypeptide. Thus,
structural data will ultimately be required to provide a definitive basis for proper
interpretation of the results obtained with the mutant polypeptide. It should be noted,
however, that our previous documentation that Gall can independently reconstitute
splicing activity in NE depleted of the galectins provided an activity of the protein that
can be assayed without measuring carbohydrate-binding (5). Our present results showing
that GST-Gall(N46D) can reconstitute splicing despite being deficient in carbohydrate-
binding suggest that we have been able to dissociate the two activities in the N46D
mutant. This, in turn, suggests that the mutant polypeptide must have retained sufficient
structure to preserve the splicing activity while the carbohydrate-binding activity was
compromised. We draw analogy to two previously documented examples. Prostaglandin
endoperoxide synthase, which catalyzes the committed step in the synthesis of
prostaglandins and thromboxanes, exhibits two activities: (a) cyclooxygenase activity;
and (b) peroxidase activity. A site-directed mutant (Tyr385Phe) lacked cyclooxygenase
activity but retained peroxidase activity, arguing against the notion that the single amino
acid substitution resulted in gross misfolding of the polypeptide (27). Similarly, Bicoid,
the anterior determinant of Drosophila, controls embryonic gene expression by
transcriptional activation and translational repression. Replacement of arginine at residue
54 (e.g. R548) shifts the binding properties of the homeodomain to prefer DNA over

RNA recognition. This abolishes mRNA translational repression without affecting

100

transcriptional activation (28). Thus, we interpret our present data to indicate that the
saccharide-binding activity is not required for the splicing activity.

We have taken advantage of this property of the N46D mutant, loss of
carbohydrate-binding activity but retention of splicing activity, to overcome the
inhibition of splicing observed in complete (non-depleted) NE containing TDG. This
raises the question of how to reconcile the two apparently disparate findings: while
carbohydrate-binding is not required for splicing activity, saccharides such as Lac and
TDG nevertheless exert an inhibitory effect when added to the splicing assay. It is
possible that binding of saccharide ligands to the carbohydrate-binding site results in a
conformational change that disrupts the interaction of the galectin polypeptide with a
component of the splicing machinery. Endogenous Gall and Ga13 in the NE would be
sensitive to TDG because they can bind to the saccharide. Likewise, exogenously added
GST-Gall(WT) cannot overcome the TDG inhibition because it also binds to the
carbohydrate ligand. In contrast, because GST-Gall(N46D) is deficient in carbohydrate-
binding activity, it will not undergo the conformational change and therefore, can retain
its functional splicing interactions.

The finding that Gall association with H splicing complexes is sensitive to
saccharide dissociation is also very interesting. In this instance, the site is accessible to
galectin-specific ligands and binding alters the overall conformation of the galectin which
prohibits other interactions. The transition from H to E and active spliceosomes causes
rearrangements that now cause the carbohydrate-binding site to be inaccessible to TDG

and resistant to release from the splicing complexes.

101

To the best of our knowledge, a conformational change in the Gall polypeptide
upon saccharide binding has not been reported. Moreover, most of the crystallographic
structures of the galectin family of polypeptides have been determined in complexes
containing saccharides (23-26). The lone exception is that of galectin-7, in which the X-
ray structure was determined in both free and carbohydrate-bound forms (29). A
comparison of the three-dimensional structures of the two forms showed that the
polypeptide does not undergo any significant conformational change upon carbohydrate-
binding. On the other hand, evidence for a conformational change in the COOH-terminal
carbohydrate recognition domain of Ga13 upon Lac binding has been suggested by
differential scanning calorimetry studies (30). In addition, several studies have
documented that Ga13 binds to multivalent ligands with positive cooperativity (31-33)
and it was suggested that saccharide-binding might expose hydrophobic surfaces for
interactions between the carbohydrate recognition domains of separate Gal3 molecules
(34). Thus, a rigorous test of our hypothesis must await physico-chemical studies on the
Gall polypeptide itself, as well as tests of the effect of Lac/TDG on the interaction

between Gall (and Ga13) with components of the splicing machinery.

FOOTNOTES
This work was supported by grants MCB 97-23615 from the National Science
Foundation (RJP) and GM-38740 from the National Institutes of Health (JLW). Patty
Voss contributed part of the work.
Abbreviations: Gall, galectin-1; Gal3, galectin-3; NE, nuclear extract; Lac, lactose;

SMN, survival of motor neuron; snRNP, small nuclear ribonucleoprotein; TDG,

102

thiodigalactoside; ASF, asialofetuin; GST, glutathione S-transferase; WT, wild-type; PBS,

phosphate-buffered saline

103

10.

11.

12.

l3.

14.

15.

l6.

17.

18.

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Chapter 4

Nuclear galectin containing complexes

Introduction

The sedimentation of galectin-3 with hnRNP and snRNP complexes in cesium
chloride buoyant density gradients is an interesting finding because it was performed
without adding exogenous pre-mRNA. This leads to a hypothesis that the galectins might
associate with nuclear complexes other than the spliceosome. To prove this hypothesis,
several experiments were carried out and the existence of such complexes was confirmed.
Some interesting properties of these complexes have also been observed in our

experiments.

Observations

A. Galectin-1 and galectin-3 associate with nuclear complexes which contain
snRNPs.

Using antibodies against galectin-1 and -3, snRNAs of U1, U2, U4, U5 and U6
were precipitated from HeLa cell nuclear extract. The procedure is similar to that
described in Chapter 2. HeLa nuclear extract was incubated at 30°C in the presence of
ATP for 30 minutes to remove any pre-existing splicing complexes. The extract was then

incubated at 4°C with antibody coupled protein-A beads for 1 hour, followed with

106

washing and 5xSDS sample buffer elution. The bound fraction was then subjected to
denaturing gel separation and resolved RNAs were transferred to hybond-N nylon
membrane using standard northern blotting transfer procedure. To reveal the RNAs after
transfer, we use both riboprobes transcribed from snRNA clones and 32p-labeled oligo
nucleotide probes as following:
U1: TCC CCT GCC AGG TAA GTA TC
U2: TTA GCC AAA AGG CCG AGA AGC GAT
U4: GGG GTA TTG GGA AAA GTT TTC
U5: ATC TGA AGA CAA ACC AGA GTA T
U6: TTC TCT GTA TCG TTC CAA T

The hybridization conditions are: using 20 ml hybridization solution [containing
3ml 20xSSC solution, 2 ml 50x Denhart’s solution, 1 ml 1M NaHzPO4,0.2 ml 10%SDS,
0.2 ml herring sperm DNA and 3 ml (for oligo probe) or 10 ml (for riboprobe)
fonnamide], hybridize membrane at 42 °C over night and wash with 2xSSC solution for 1
houn

Figure 1 shows the antibody precipitated snRNAs after northern blotting. As we
expected, pre-immune serum only precipitated background levels of snRNAs, while
significant amounts of snRNAs were precipitated by anti-galectin-l and anti-galectin-3.
These data strongly indicate the presence of galectin-1 and galectin-3 in snRNP

containing complexes.

107

Figure 1: Galectin-1 and galectin-3 associate with nuclear complexes
which contain snRNPs and this association is sensitive to ionic strength.
In panel A, using antibodies against galectin-1 and -3, snRNAs of U1,
U2, U4, U5 and U6 were precipitated from HeLa cell nuclear extract.
Lane 1 shows nuclear snRNAs (U1, U2, U4, U5 and U6) as positive
control. In lane 2, preimmune serum only precipitates background level
snRNPs. In lanes 3 and 4 anti-galectin-l and -3 precipitate snRNAs,
respectively. Lanes 5 and 6 show similar precipitation under high ionic
strength condition (200mM KCl). Note the amount of snRNAs
precipitated by galectin antibody was greatly reduced under such
condition. Panel B shows the western blotting of galectin-3 in the bound
fraction with the same lane order.

108

Figure l

60mM KCL 200mM KCL

NE 1" agaH agal3 Pl 093.3

"‘ 13")“ . .2...”
.

    

‘.‘~y..»,l~.aiw ‘ .12.;11' ‘

illll. V .1. ‘llllllliiiilii

,;;;ljllll'-

l .

109

U2
U1

U4

U5

U6

' aMac2

B. The association of galectins with nuclear complexes is sensitive to ionic strength.

Since ionic strength is a very important factor in galectins’ association with
spliceosomes, it was interesting to see how ionic conditions affect the association of
galectins with nuclear complexes. We used the same method for immunoprecipitation,
but increased the salt concentration of the binding and washing solutions from 60mM to
200mM. As shown in Figure 1, increasing the salt concentration significantly reduced the
amount of RNA that precipitated. This suggests that galectin-1 and -3 probably
incorporate into different complexes (i.e. spliceosomes and snRNP containing complexes)

through a similar mechanism.

C. The association of galectins with nuclear complexes is not affected by

carbohydrate ligands.

In previous experiments testing carbohydrate ligands effect on galectins’
interaction with spliceosomes, TDG inhibited the precipitation of pre-mRNA in H
complex, but not in E and active splicing complexes, indicating the association of
galectins with the active spliceosomes can not be disrupted by carbohydrate ligands. A
similar experiment performed without pre-mRNA will give us a clue of the role of the
CRD in galectins’ association with nuclear complexes. HeLa nuclear extract was pre-
incubated with 200mM TDG or cellobiose. Splicing reaction has been documented to be
inhibitable by TDG at this concentration. Then the nuclear extract was subjected to anti-

galectin-l immunoprecipitation. Figure 2 shows the effect of TDG on anti-galectin-l

110

Figure 2: The association of galectins with nuclear complexes is
not affected by carbohydrate ligands. HeLa nuclear extract was
pre-incubated with 200mM TDG or cellobiose. Then the nuclear
extract was subjected to anti-galectin-l immunoprecipitation.
Comparing to regular precipitation (lane 4), there were no
significant changes in the amount of snRNAs precipitated with
the addition of TDG (lane 5) or cellobiose control (lane 6).
Preimmune (lane 2) and anti-Sm (lane 3) served as negative and
positive controls.

111

Figure 2

NE Pl

    

iii“ IIII‘II'III

1

III lllllllllllllIlIlllllllll i“

IIIIIIIIIIIIIIII. IIIIIIIIII

2

aSm agal1 agal1 agal1

112

+TDG +cello

U2

U1

U4

U5

U6

precipitating snRNAs. There is no change in the quantity of snRNAs precipitated with the

addition of TDG into the extract when comparing lanes 4, 5 and 6.

D. Galectin-1 and -3 are also mutually exclusive in their association with nuclear

complexes.

We previously discovered that the association of galectin-1 and -3 with
spliceosomes is mutually exclusive, indicating there are at least two different types of
spliceosomes. This raises the question whether galectin-1 and -3 associate with snRNP
containing nuclear complexes in a coordinate or an independent way. To address this
question, we took advantage of the sequential immunoprecipitation method. Using the
procedure described in Chapter 2, nuclear extract was subjected to anti-galectin-l and
anti-galectin-3 in a sequential manner, the bound and unbound fraction from each
precipitation was collected and separated by gel electrophoresis. The snRNA and protein
components were analyzed to determine the nature of the associations. As shown in
Figure 3, in the first immunoprecipitation, preimmune serum did not precipitate any
snRNAs. Anti-Sm precipitated snRNAs and served as a positive control. Anti-galectin-l
and anti-galectin-3 both precipitated snRNAs in this round as they did in previous
experiments. When the unbound fraction of the first precipitation passed through the
opposite antibody beads in the second round of precipitation, significant amounts of
snRNAs were still precipitable. The western blot data shows that after extract went
through the first antibody column, >85% of the galectin-l or galectin-3 protein was

removed from the extract, but the opposite protein was still present in the first

113

Figure 3: Galectin-1 and -3 are mutually exclusive in their association with
nuclear complexes. HeLa nuclear extract was subjected to anti-galectin-l
and -3 in a sequential manner, the bound and unbound fraction fiom each
precipitation was collected and the snRNA and protein components were
analyzed, respectively. Panel A shows the snRNAs precipitation. In the
first immunoprecipitation, anti-galectin-l and anti-galectin-3 both
precipitated snRNAs from the nuclear extract (lanes 4 and 5). Preimmune
(lane 2) and anti-Sm (lane 3) served as negative and positive controls.
When the unbound fraction of the first precipitation passed through the
opposite antibody in the second precipitation, significant amounts of
snRNAs were still precipitable (lanes 7 and 8). Panel B shows the western
blotting (aMac2 was used to detect galectin-3) of the unbound fraction of
the first precipitation. Lanes 6 and 7 shows >85% of the galectin-1 or
galectin-3 protein was removed from the extract by its own antibody, but
the opposite galectin was still present.

114

Figure 3

A lst "3 2nd IP

NE Pl aSm agal1agalL NE agal1aLal3

mmmmt‘

H
“I.
‘uumw

 

i aMacZ

 

agal1

115

unbound fraction, indicating the removal of one galectin does not affect the other. These
data suggest that each galectin associates with a subset of snRNPs independently of one

another.

Discussion

In this series of experiments, we got the following major results: 1, Galectin-1 and
galectin-3 associate with nuclear complexes which contain snRNPs; 2, The association of
galectins with nuclear complexes is sensitive to ionic strength; 3, The association of
galectins with nuclear complexes is not affected by carbohydrate ligands; 4, Galectin-1
and -3 are also mutually exclusive in their association with these nuclear complexes.

Most of these findings are consistent with the behavior of galectin-1 and -3 in the
spliceosomes. These results could be predicted because it is easy to conceive that
galectin-l and -3 are more likely to be a component of pre-existing splicing related
complexes than a free protein in the nuclear extract. Dreyfuss’ study has indicated that
nuclear snRNP particles go through regenerating cycles. Nuclear galectins are likely to be
involved in such process. They probably incorporate into the particles and, as we have
suggested, deliver the snRNPs to the spliceosomes. The precipitation of snRNAs from
nuclear extract by galectin antibodies strongly supports this idea.

Like the association with spliceosomes, the association of galectins with nuclear
complexes is sensitive to ionic strength. This suggests galectins probably loosely bind to
some protein components in the nuclear complex. A weak interaction does not mean it is

an unimportant one. In fact, many regulatory factors bind to their binding partners with a

116

 

rather “weak” interaction. Galectin-3 has been suggested to be a regulatory factor in other
research. In thyroid cancer cell, galectin-3 directly interacts with a transcription factor
TTF-l, stimulates the DNA binding activity of this protein and thus contributes to the
proliferation of the thyroid cell. Galectin-3 has also been reported to interact with Sufu
protein, which is a negative regulator of the Hedgehog signal transduction pathway.
Galectin-3 can affect the localization of this protein between nucleus and cytoplasm and
hence play a role in regulating this specific pathway. Overall, the role of galectins in
splicing maybe far more sophisticated than we expected.

The fact that TDG did not affect galectins’ association with the snRNPs is not
unexpected. In previous studies, we have found out that adding excess TDG can only
affect the association of galectins with the H complex, but not that of galectins with the E
and active splicing complexes. We have proposed that when TDG binds to galectin-1 or -
3, it will induce a conformational change and thus prohibit galectins from binding to their
protein partners in the splicing complex. This is probably the case of the H complex. On
the other hand, in the nuclear complexes or active splicing complexes, because galectins
have incorporated stably into a complex, it will be covered by other proteins and won’t be
accessible to TDG anymore. Hence TDG won’t affect the association of galectins with
these complexes.

The sequential precipitation study proved our theory that in the nuclear extract,
there are pre-existing galectin containing snRNP complexes and galectin-l and -3 are
mutually exclusive in these complexes (i.e. one complex only containing one type of
galectin). When pre-mRNA is added to the extract, galectins might facilitate the

complexes they associated with to interact with the pre-mRNA, deliver the necessary

117

 

factors to the splicing reaction. This finding is consistent with galectin-spliceosome
association pattern. Since galectin-1 and -3 do not coexist in the pre-existing complexes,

they would not associate with the spliceosome in a co-existing manner.

118

Chapter 5

Conclusion and future plans

Pre-mRNA splicing is a very important post-transcriptional process mediated by a
large complex called the spliceosome. Previous studies have confirmed that nuclear
galectin-1 and -3 are essential splicing factors. After years of working on galectins and
splicing, we have achieved some important discoveries and elucidated part of the roles of
galectins in the splicing process.

My work has partially solved the problem of how galectin-l and -3 exert their
functions in the splicing process. Galectin-1 and -3 are associated with nuclear snRNP
containing complexes without pre-mRNA. After pre-mRNA is made, galectins will
facilitate the delivery of the snRNP particles onto the pre-mRNA which allowed the
sequential forming of the E, A, B and C splicing complexes. The fimction of galectins
starts at the assembly of the B complex and they may slowly dissociate from the
spliceosomes during the splicing process (Figure 1). Each of the nuclear galectins
functions by itself and excludes the other from the same complex.

There are still many questions need to be addressed concerning galectins’ roles in
splicing. We need to isolate the nuclear galectin containing complexes and determine the
constituents of these complexes. Silver staining of polyacrylamide gels, mass
spectrometry and database searches could be used to enumerate and identify the
polypeptides of these complexes. This could give us clues to determine the real binding
partners of galectins in the splicing process and further elucidate the mechanism of

splicing. Whether galectins have other important roles in the spliceosome after they

119

Figure 1: Schematic diagram illustrating the role of galectin-1 and
-3 in the splicing process. Galectin-l and -3 are associated with
nuclear snRNP containing complexes without pre-mRNA. Each of
such complexes contains only one type of galectin. After pre-
mRNA is made, galectins facilitate the delivery of the snRNP
particles onto the pre-mRNA which allow the sequential forming
the E, A, B and C splicing complexes. When mature mRNA was
released, galectins might be involved in the regeneration process of
the snRNPs.

120

Figure 1

Working Model

spliceosome Nuclear galectin

® containing complexes
- A'-
H

 

 

121

delivering the snRNPs is another important question. Many splicing factors are involved
in spliceosomal protein and RNA rearrangement and relocation. Do nuclear galectins also
have such role in these processes? Lastly, what happens after the mRNA is released and
the spliceosome dissociates? We have seen data suggesting that galectins could be
involved in the regeneration of snRNP particles in the nucleus. This should be a new area
for us to explore.

Another line of my research has discovered that the carbohydrate binding activity
of galectins is separated from their splicing activity. The evidence is that mutant galectins
without carbohydrate binding activity can still reconstitute splicing reaction in a galectin
depleted system. To further confirm this conclusion, we should construct a mutant
galectin which abolished its splicing activity and test its ability to bind to lactose or TDG.
A positive result of such experiments will strengthen our conclusion drastically. It is also
important to find out the entire conformational changes of galectins after they bind to
lactose. It will help us to understand why lactose can inhibit splicing and determine the
binding surface of galectins with their partners in the spliceosome.

The data contained in this thesis have laid a foundation for these future studies.

The entire role of nuclear galectins in splicing will be eventually determined.

122

   

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