.. .‘ V ,.:y e 3 , . 1 h $1.?dmu adatnm y I V33} (new. 2. {tit‘m he}??? .5 r LTD. .\. 5.1., E... 18%? . P , . Li, unxfuwimfl» 3.. _. i . » 74“.;‘JPVE. 2' t 1.1 (.31 4 .l ‘3'|‘£t}“.‘o~ I- I I ‘ .u).\itn£~1§ I. .... a. .3 .. . ) 92 lflhuiu mun. .. up}: ... .2. !. in... a 2. Ant. . o. 3. .. 31.x»... :s t. r. 1.. 5.0.0:}? 1|! 3.1.1..» tit. .5 . an (a! f 5.»: in . 3.51.11. -. . 5:. . I x . .11: . s. 3 lh11i\ 2... i “‘v’. . ban 3 sii...t..a.?- .2211: :3; iii. t. 534.1 Eiflml...‘ dump! I’M}: .t\ut1’ i 1C) LIBRARY Michiga; State Universnty This is to certify that the dissertation entitled INTERACTION OF DICER, TRBP WITH SHORT INTERFERING RNAs: EFFECT ON SILENCING EFFICACY OF siRNAs presented by HEMANT K. KlNl has been accepted towards fulfillment of the requirements for the Ph.D degree in Chemical Engineering 4% ’ Major Professor’s Signature // 30/27 Date MSU is an Afiinnative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:/Proi1Acc&Pres/CIRC/DateDuo.indd INTERACTION OF DICER, TRBP WITH SHORT IN TERFERIN G RNAs: EFFECT ON SILENCING EFFICACY OF siRNAs By HEMANT K. KINI A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chemical Engineering 2009 ABSTRACT INTERACTION OF DICER, TRBP WITH SHORT INTERFERING RNAs: EFFECT ON SILENCING EFFICACY OF siRNAs By HEMANT K. KINI Gene silencing by RNA interference (RNAi) is mediated by endogenous proteins and leads to target mRNA cleavage or translational inhibition. In the cytoplasm, the dsRNA binding proteins, TRBP and Dicer, recognize and bind the siRNA, and form the RNA induced silencing complex (RISC) loading complex (RLC). Argonaute 2, which forms the catalytic core of RISC, is then recruited to this protein complex. Ag02 then gets loaded with the guide strand, the strand complementary to the mRNA, and silences the target mRNA. In this research, a novel role for human Dicer has been demonstrated. It was found that Dicer binds 21-nt ssRNAs, having higher affinity for those possessing a 5'-phosphate relative to a 5'-hydroxyl. Using liquid chromatography-mass spectrometry (LC/MS), the different Dicer domains binding the ssRNAs vs. the double stranded siRNAs were preliminarily identified. Based on these findings and prior findings, a model has been proposed for the loading of Ag02 with the single stranded guide strand. Dicer along with TRBP recognizes, binds with the siRNAs and forms the RLC. This constitutes an important step in the RNAi pathway. To enhance silencing and avoid off target effects, siRNAs are often designed with an intentional bias to make the end of the siRNA containing the guide strand 5'-end less stably hybridized relative to the end containing the passenger strand 5'-end. By studying a number of siRNAs with terminal mismatches, it was found that siRNA-TRBP binding is largely indicative of eventual silencing efficacy of the siRNAs and that this binding can be significantly reduced by terminal mismatches. TRBP-siRNA complex formation is more tolerant of internal mismatches than terminal mismatches. Terminal mismatches did however lead to a small increase in Dicer binding, though without a concomitant improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always serve to improve target silencing and that care should be taken when designing siRNAs in this way. Here, a previously unknown function of Dicer protein in its ability to bind ssRNAs, and the impact of TRBP protein on the silencing activity of asymmetric siRNAs with a terminal mismatch has been demonstrated. Overall this research has furthered the knowledge of the functional roles of two important proteins, Dicer and TRBP, in the RNAi pathway. Copyright by Hemant K. Kini 2009 To my parents. ACKNOWLEDGEMENTS I am grateful to several people for their help, support, friendship, and guidance over these years as a graduate student. First and foremost I would like to offer my gratitude to my advisor, Pat Walton whose constant support and guidance was crucial to me and my research. I am thankful to Pat for not only inspiring me to think independently, but also giving me the freedom to plan and direct my own research projects. I would like to offer my special thanks to all the members of my Ph.D committee (Dr. Donna Koslowsky, Dr. Kris Chan, and Dr. Charles Petty) for their valuable insights and suggestions. To Dr. Kris Chan for her positive critique of my work, words of encouragement and insightful suggestions. Dr. Charles Petty who was also my masters thesis advisor, for getting me started on the path of scientific research. I am truly'grateful to the members of the Proteomics core (Doug Whitten) and the Mass spectrometry facility (Dr. Dan Jones and Dr. Lijun Chen) at MSU, for the training and ungrudging help they have provided me. Particularly to Dr. Dan Jones and Dr. Lijun Chen, who personally helped me with several of my mass spectrometry experiments and data analysis, despite their hectic schedules. I would also like to extend my special thanks to all the members of Chan and Walton labs, who were a great team to be part of. To Shireesh, Sheenu, Sachin, Sumit, Joe, Xuerui, Linxia, Hirosha, and Shengnan for their collaboration, support and camaraderie. I would also like to thank the Chemical Engineering and Materials Science Department, the College of Engineering and, Office of International Students and Scholars (0188) for their financial support. In particular, I would like to thank the staff from the Chemical vi Engineering and Materials Science Department, Nancy, JoAnn and Eunice for their administrative support with cheerful demeanor. To my dear friends who always made the Windy city a warm enclave for me Sandeep Gaonkar, Raj Naik, and rest of the Gaonkar, Naik and Torke clan. To Mrs. Marina Farhat, Gina Farhat, and Susan Farhat who always treated me like family. Gratzi. To all the friends to whom I am thankful for the good times, great memories, support and sage advice Afshan (Bawa), Bhavin, Sharad, Madhu, Srivatsan, Krishnan, Amit, Harish and Chetan. Family and friends have been the quintessential rock of support for me through all these years. My utmost gratitude to my parents for their love, support and sacrifices they had to make, for me to be able to get here. To my brother, Ajay for not only for giving me a cute nephew (Tanay), but also for all his unerring love and support. Most importantly to my wife Akshata, for her love, understanding, support and trust which I can always rely on. I appreciate it more than you will ever know- thank you. vii TABLE OF CONTENTS LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii CHAPTER I 1 RNAi pathway: initiation to application 1 INTRODUCTION 1 RNA interference (RN Ai) ........................................................................................... 1 Initiation of RNAi: Dicer, miRNAs and siRNAs ........................................................ 1 Initiation complex - RLC ............................................................................................. 3 Active RISC and silencing of target mRNA ............................................................... 7 RNAi : discovery to application ................................................................................ 10 Applications of RNAi in biotechnology and biomedicine ........................................ 10 Summary, aims, and findings of this research ........................................................... 14 CHAPTER II 17 Human Dicer binding of ssRNAs and siRNAs 17 INTRODUCTION 17 RESULTS AND DISCUSSION 20 SsRNA-Dicer complex formation in vitro ................................................................ 20 Possible contribution of the PAZ domain to ssRNA binding by Dicer ..................... 23 5' terminal nucleotide sequence dependence of Dicer binding ................................. 26 Divalent cation dependence of ssRNA-Dicer complex formation ............................ 29 SsRNA binding by Giardia Dicer ............................................................................. 31 Identifying the Dicer domain/s involved in binding with the ssRNA and siRNAs... 33 Identifying the Dicer domains binding siRNA and ssRNA by comparison of the triple spectra .............................................................................................................. 38 CONCLUSIONS 43 PAZ domain mediated ssRNA and siRNA binding by Dicer ................................... 43 Divalent cation dependence of ssRNA—Dicer complex ............................................. 44 Dicer domains stabilizing binding with ssRNAs and siRNAs .................................. 45 Proposed model for Dicer-ssRNA binding and Ag02-Dicer interaction ................... 46 CHAPTER III 50 Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy 50 INTRODUCTION 50 RESULTS 52 Design of siRNAs and EGFP silencing efficiency .................................................... 52 Effect of guide strand 5'-end mismatch on TRBP and Dicer binding ....................... 57 Effect of siRNA structure and composition on siRNA protein complexes ............... 63 CONCLUSIONS 66 viii TRBP, Dicer binding of siRNAs and functionality of the siRNAs ........................... 66 CHAPTER IV 68 Summary and future directions - _ - 68 Major contributions of this research .......................................................................... 68 Future Work .......... . .................................................................................................... 69 Characterizing other RNAi pathway complexes in cell extracts ............................... 69 Is it RN Ai ? ................................................................................................................ 7O siRNAs with terminal and internal modifications ..................................................... 71 Binding studies with Dicer mutants .......................................................................... 71 APPENDIX A 72 Characterizing Dicer protein preparation .................................................................. 72 Mass spectrometry analysis of unknown complexes formed by ssRNAs ................. 73 Generating theoretical cleavage map for tryptic digest of a protein ......................... 74 Methods and materials ............................................................................................... 78 APPENDIX B -- -- 81 Terminal stability analysis of siRNAs with terminal mismatches ............................ 87 Calculating terminal stability of the siRNAs ............................................................. 89 Methods and materials ............................................................................................... 92 .............. -- 97 REFERENCES... -_ - ...... ix LIST OF TABLES Table 2.] Predicted AG and TM for the sequences predicted using mfold ........................ 23 Table 2.2 Predicted AG and TM for the sequences predicted using mfold ........................ 28 Table 2.3 Amino acid sequence of human Dicer domains ............................................... 40 Table 3.1 Difference in end stabilities of the siRNAs ...................................................... 53 Table Bl SiRNAs with terminal modifications (Ding et al. 2007) ................................. 87 Table B2 Sequence of siRNAs used to target EGFP ....................................................... 88 Table B3 siRNAs with terminal modifications (Holen et al. 2005) ................................. 89 LIST OF FIGURES Figure 1.1 RNase III activity of Dicer. ............................................................................... 3 Figure 1.2 Formation of RLC and hole-RISC .................................................................... 5 Figure 1.3 Relative stabilities of 5' ends determines the guide strand of a siRNA ............. 7 Figure 1.4 Transition from holo—RISC to active RISC ....................................................... 8 Figure 1.5 Target mRNA silencing by active RISC. .......................................................... 9 Figure 2.1 Representation of the various domains of RNase 111 family proteins. ............ 19 Figure 2.2 In vitro binding of Dicer with siRNA and ssRNAs ......................................... 21 Figure 2.3 Impact of 5'-phosphate on Dicer binding affinity for ssRNAs and siRNAs. .. 24 Figure 2.4 Dicer has lower affinity for ssRNAs having a 3'-biotin. ................................. 27 Figure 2.5 Dicer ssRNA binding is dependent on the terminal nucleotide sequence. ...... 28 Figure 2.6 Divalent cation dependence of Dicer-ssRNA complex formation. ................. 30 Figure 2.7 Schematic of the Giardia Dicer domain and its in vitro binding with ssRNAs. ........................................................................................................................................... 32 Figure 2.8 Sequence of steps to determine the Dicer domain/s binding with the ssRNA and siRNA ......................................................................................................................... 34 Figure 2.9 LC/MS chromatograms ................................................................................... 37 Figure 2.10 Overlaid spectra of proteolysed Dicer, Dicer-siRNA and Dicer-ssRNA. ..... 40 Figure 2.11 Overlaid spectra of proteolysed Dicer, Dicer-siRNA and Dicer-ssRNA. 42 Figure 2.12 Schematic of Dicer interaction with siRNAs and ssRNAs ........................... 48 Figure 2.13 Schematic of Ag02 loading with the guide strand ......................................... 49 Figure 3.1 Effect of guide strand 5'-end mismatch on silencing efficacy of siRNAs ....... 54 Figure 3.2 Effect of TRBP or Dicer knockdown on the silencing efficacy of the EGFP targeting siRNAs ............................................................................................................... 56 Figure 3.3 Characterizing siRNA-TRBP and siRNA-Dicer complexes ........................... 58 xi Figure 3.4 Characterizing Dicer, TRBP compmlexes in H1299 cell extracts .................. 60 Figure 3.5 Effect of terminal mismatch at guide strand 5'-end on siRNA-TRBP and siRNA-Dicer complex formation ...................................................................................... 61 Figure 3.6 Effect of terminal and internal mismatches on siRNA-TRBP and siRNA-Dicer complexes. ........................................................................................................................ 64 Figure Al Characterizing ssRNA-protein complexes ...................................................... 72 Figure A2 Mass spectrometry of the faster moving complexes formed by the ssRNA (denoted by the dotted arrows in Figure 2.6). ................................................................... 73 Figure A3 Spectra of trypsinized BSA protein ................................................................. 75 Figure A4 Spectra of trypsinized BSA protein ................................................................. 76 Figure A5 LC/MS chromatograms ................................................................................... 77 Figure B1 EGFP silencing efficacy of siRNAs at different concentrations .................... 81 Figure B2 Western blot analysis of TRBP and Dicer levels in H1299 cells. .................. 82 Figure B3 Characterization of siRNA-TRBP complex formation after TRBP knockdowrgr3 Figure B4 Effect of a terminal mismatch at the guide strand 5' end on siRNA-TRBP complex formation. ........................................................................................................... 84 Figure B5 Effect of a terminal mismatch at the guide strand 5' end on siRNA-TRBP complex formation. ........................................................................................................... 85 Figure B6 Structure of siRNAs with terminal and internal mismatches ......................... 86 Figure B7 Structure of siRNA. ........................................................................................ 90 Figure B8 Mfold 2 state hybridization server webpage listing thermodynamic details. . 91 xii Ag02 ATP Bi BSA Da Dcr DNA dsRBD dsRNA DUF283 EDTA eIF4E EGF P FBS HeLa HepG2 H 1299 HIV kDa Loqs miRNA mRNA LIST OF ABBREVIATIONS Argonaute 2 Adenosine triphosphate Biotin Bovine serum albumin Daltons Dicer Deoxyribonucleic acid Double-stranded RNA binding domain Double-stranded RNA Domain of unknown function 283 Ethylenediaminetetraacetic acid Eukaryotic translation initiation factor 4E Enhanced green fluorescent protein Fetal bovine serum Human cervical carcinoma cell line Hepatocellular carcinoma cell line Non-small cell lung carcinoma cell line Human immunodeficiency virus kiloDalton Loquacious mass to charge ratio of peptides microRNA Messenger RNA xiii nt Nucleotide PACT PKR-activating protein PAZ Piwi Argonaute Zwille PBS Phosphate buffered saline PKR Protein kinase R R2D2 Protein with two RNA domains (R2) and interacting with Dicer 2 (D2) RISC RNA-induced silencing complex RLC RISC-loading complex RNA Ribonucleic acid RNAi RNA interference RNase Ribonuclease shRNA Short hairpin RNA siRNA Short interfering RNA ssRNA Single-stranded RNA TAR Transactivating response TBE Tris-borate-EDTA TRBP TAR RNA binding protein UV Ultraviolet xiv CHAPTER I RNAi pathway: initiation to application INTRODUCTION RNA interference (RNAi) RNA interference (RNAi) is an evolutionarily conserved mode of gene regulation and host defense in eukaryotes (Cullen 2002; He and Harmon 2004). Short interfering RNAs (siRNAs) can be designed to specifically target and regulate the expression of any particular gene. However dsRNAs longer than 30bp activate the mammalian cell defense mechanism leading to induction of cytokine production and cell death (Williams 1999; Caplen et al. 2001; Elbashir et al. 2001a). Hence, 2l-mer siRNAs are the most commonly used silencing agents for mammalian systems (Elbashir et al. 2001a). SiRNAs have been widely employed as tools to study gene fiinction, regulating protein expression, and in therapeutic applications (Dorsett and Tuschl 2004; Whitehead et al. 2009). SiRNAs are similar in structure to endogenously encoded microRNAs (miRNAs), which regulate diverse cellular pathways and are key regulators of development and disease (Stefani and Slack 2008; Ghildiyal and Zamore 2009). Both of these active species share similar components and mechanisms of the RNAi pathway. Initiation of RNAi: Dicer, miRNAs and siRNAs RNAi can be initiated by exogenous or endogenous means. In endogenous regulation, primary miRNAs (pri-miRNAs) are produced in the nucleus by RNA polymerases II or III. These are then processed by the nuclear complex of Drosha, an RNase III family enzyme, and its dsRNA binding partner, which is known as Pasha in Drosophila (Denli et al. 2004) and DGCR8 in humans (Gregory et al. 2004; Han et al. 2004). This processing yields precursor miRNAs (pre-miRNAs) (Lee et al. 2002; Zeng and Cullen 2003). Pre- miRNAs have 5'-phosphates and the 3' dinulceotide overhangs characteristic of RNase III processing (Basyuk et al. 2003; Lee et al. 2003). Once generated, pre-miRNAs are exported to the cytoplasm by Exportin-S-Ran-GTP (Y i et al. 2003). In the cytoplasm, Dicer, another RNase III family enzyme, further processes the pre-miRNA to the ~21- mer mature miRNAs (Lee et al. 2003). In a similar manner, Dicer can process any long dsRNA substrate to siRNAs with similar structure to miRNAs. Human Dicer, which generates both siRNAs and miRNAs (Fig. 1.1), is a multi-domain protein with an N-terminal RNA helicase/ATPase domain, a domain of unknown function (DUF 283), a Piwi Argonaute Zwille (PAZ) domain, two RNase 111 domains (RIIIa and RIIIb), and a dsRNA binding domain (dsRBD) (Hammond 2005; Cook and Conti 2006). The RNase domains cleave the target dsRNA into siRNAs and miRNAs with characteristic 2 nt overhangs at each 3' end (Lee et al. 2002; Zhang et al. 2004). The C-terminal dsRBD and the PAZ domain orient the dsRNA for cleavage at the proper locations (Zhang et al. 2004). Drosophila has two Dicers, Dcrl and Dcr2, which have distinct roles in the miRNA and siRNA pathways (Lee et al. 2004; Farazi et al. 2008). Dcrl requires the dsRNA binding protein Loquacious (Loqs) for processing of pre—miRNAs into mature miRNAs (Forstemann et al. 2005). Unlike Dcrl, Dcr2 alone can cleave long dsRNA substrates (Forstemann et a1. 2005). Similar to the Drosophila Dicers, the human Dicer is also associated with a partner protein, the human immunodeficiency virus (HIV-l) transactivating response (TAR) RNA-binding protein (TRBP) (Chendrimada et a1. 2005; Haase et a1. 2005). Dicer processing of long dsRNA Dicer processing of pre-miRNA Dicer ‘ " Dicer Dicer —siRNA complex Dicer —miRNA complex e” Dicerl||ll|l|I||||||||||||| ’ Diceriiili lllllll llll III _lJiLllLLllllJJJJ.LlJJJJ lllllll “III“ III II Figlre 1.1 RNase III activig of Dicer. Dicer processes long dsRNA or pre-miRNA substrates into siRNAs and miRNAs, respectively. Dicer is suggested to stay bound with the siRNAs/miRNAs it generates and facilitate their incorporation into RISC. Initiation complex - RLC Dicer generated siRNAs and miRNAs have a 5'-phosphate and 3'-hydroxyl (Zhang et a1. 2002). Exogenously introduced, chemically synthesized siRNAs, which have a 5'- hydroxyl, upon entry into cells, are immediately phosphorylated by the human RNA kinase hClpl (Nykanen et al. 2001; Weitzer and Martinez 2007). In the cytoplasm, siRNAs and miRNAs possessing a 5'—phosphate are then recognized by Dicer and TRBP to form the RNA induced silencing complex (RISC) loading complex (RLC). TRBP TRBP is a dsRNA binding protein known to stimulate the expression of HIV-1 virus (Duarte et al. 2000; Gatignol and Jeang 2000; Daher et al. 2001) and inhibit the activity of another dsRNA binding protein kinase R (PKR) (Daher et al. 2001). TRBP has two dsRNA binding domains and the C-terminal Medipal domain, which interacts with the other proteins, namely Merlin, Dicer, and PKR activating protein, to which TRBP is structurally similar (PACT) (Laraki et al. 2008). However, they exert opposite regulatory effects on PKR, with PACT activating PKR (Patel and Sen 1998; Li et al. 2006) and T RBP inhibiting PKR (Daher et al. 2001). TRBP interacts with Dicer in processing pre-miRNAs and also in forming the RLC (Fig. 1.2) (Chendrimada et al. 2005; Haase et al. 2005). The RLC then recruits Argonaute 2 (Ag02) to form holo—RISC (Fig. 1.2). RISC catalytic protein - Argonaute 2 (Ag02) Ag02 belongs to the Argonaute protein family which derives its name from typical squid- like phenotype witnessed in plants lacking Argonaute proteins (Peters and Meister 2007). These proteins are characterized by the presence of PAZ (Piwi-Argonaute-Zwille) and PIWI (P-element induced wimpy testis) domains (Bohmert et al. 1998). These proteins are classified into the Ago subfamily and the Piwi subfamily (Peters and Meister 2007; Hutvagner and Simard 2008). While Ago proteins are ubiquitously expressed, Piwi proteins are restricted to germline cells (Carmell et al. 2007). Piwi proteins interact with another class of short interfering RNAs called piRNAs (Piwi-interacting RNAs) which Ag02 --—-- .... Ag02 Holo-RISC I Dicerw TRBP r Figge 1.2 Formation of RLC Ed holo-RISC Dicer and TRBP recognize siRNAs and form the RLC. Ag02 is then recruited to this complex to form holo-RISC. are necessary for normal germline development (Das et al. 2008; Klattenhoff and Theurkauf 2008). Various Argonaute proteins have been shown to be involved in diverse cellular functions from embryonic development to transposon silencing (Lykke-Andersen et a1. 2008; Siomi and Siomi 2008). The number of Argonaute genes varies between species with 27 in Celegans to 8 in humans (Peters and Meister 2007). Out of these 8 human Argonaute proteins, Ag02 is the only protein possessing endonuclease activity (Meister et al. 2004). The crystal structure of the Pyrococcus furiosus Argonaute PIWI domain shows that this domain is very similar to RNase H, supporting the role of the PIWI domain as the active center of RISC (Song et al. 2004). While RNase H cleaves the RNA in a DNA-RNA duplex, Ag02 cleaves an RNA strand of an RNA-RNA duplex. For 5 silencing, this results in cleavage of the target mRNA when hybridized to the guide strand (Liu et al. 2004; Rivas et al. 2005). Due to its RNase activity, Ag02 is also referred to as ‘Slicer’ (as a counterpart to Dicer). siRNA containing holo-RISC to guide strand programmed active RISC Minimal RISC has been reconstituted in vitro using only purified Ag02, Dicer, and TRBP (MacRae et al. 2008). Activation occurs when one strand is destroyed during the process and thereby creating an active RISC. While either strand of a siRNA duplex can be part of active RISC, chemically synthesized siRNAs are typically biased such that only one strand is preferentially contained in active RISC (Schwarz et al. 2003). The initial four base pairs at each 5' end of the siRNA duplex are typically used to determine the stability of that end (Fig. 1.3). The strand with the relatively unstable 5' end becomes the guide strand and the complementary strand becomes the passenger strand. In Drosophila, the protein R2D2 senses the more stable 5' end of the siRNA duplex and binds to that end thereby positioning Dcr2 to the less stable end of the duplex, and forming the RLC (Tomari et al. 2004). TRBP, which is the human ortholog of R2D2, might play a role in sensing relative thermodynamic bias in humans (Haase et al. 2005; Pellino 2007). Following asymmetry sensing, the RLC then positions the less stable 5' end of the siRNA ’ to anchor with the phosphate binding pocket of the Ag02 PIWI domain (Ma et al. 2005; Parker et al. 2005). Then Ag02 cleaves the passenger strand phosphodiester bond across the 10 and 11 nucleotides of the guide strand (Matranga et al. 2005; Leuschner et al. 2006) similar to the cleavage of target mRNA by the guide strand (Fig. 1.4) (Martinez et al. 2002). This mode of passenger strand cleavage works only for siRNA duplexes whose strands are perfectly base paired around the passenger strand cleavage site. For miRNAs, 6 which have internal mismatches, the programming of RISC with the guide strand is predicted to occur by a bypass mechanism which does not involve passenger strand cleavage (Matranga et al. 2005). Active RISC and silencing of target mRN A Purified Ag02, when provided double-stranded siRNAs, lacks the ability to form an active RISC programmed with the single-stranded guide strand (Rivas et al. 2005). However, Ag02 alone can combine with a ssRNA acting as a guide strand to form an active RISC (Rivas et al. 2005). In vitro experiments have shown that once Ag02 is loaded with the guide strand it dissociates from Dicer and TRBP (Fig. 1.4) (MacRae et al. 2008) U: ||||§||||l|l|llll IIIIEII’ _l__|__|__i||l||||||||| = [Hi “3 [AGGgabS < [:AGpglabs figure 1.3 RelaLtive stabilities of 5' ends determines the guide strand of aLsiRNA First four base pairs at the 5' end contribute to the stability of that end. This schematic represents a case where the 5' end of the red strand is less stable than the 5' end of the blue strand. So the red strand is becomes the guide strand (guides RISC to the target mRNA) and the blue strand is designated the passenger strand (is subsequently destroyed). Holo-RISC Holo-RISC (siRNA-Dicer-TRBP-AgoZ) (miRNA—Dicer—TRBP-AgoZ) A .. Ag02 A ._ Ag02 . m . ' . ”mm-m ‘ RWDICCW-L' TRBP Dicer-W TRBP siRNA Mechanism WWW” passenger unknown for strand gets miRN As cleaved by Ag02 A802 nnniixnigrfiiimm IITI'I'I'ITl'ITTlTlTlTI'I'I'l Active-RISC Active-RISC F_igure 1.4 Trgtsition from holo-RISC to active RISC The passenger strand of the siRNA in the holo-RISC gets cleaved by Ag02 to form the active RISC programmed with the guide strand. The mechanism of unwinding/cleaving of the passenger strand for miRNAs is currently unknown. Active RISC then binds with the target mRNA based on sequence complementarity. If there is sequence complementarity between the guide and the target around the central part of the guide strand, then Ag02 cleaves the target mRNA (Elbashir et al. 2001a; Holen et al. 2002) (Fig. 1.5). Following substrate release, active RISC is liberated and can engage in multiple cycles of target cleavage (Haley and Zamore 2004; Rivas et al. 2005) Active-RISC Ag02 / \ Ag02 Ag02 mmmmmrmm mmmmrmmm WWW Ag02 Ag02:— mtmnmttmnm IITTI'I'I'ITHITI'ITTITTI'I lJJJlllIlllJllllllJllllllJJJlllJlllJllllll llllllll|||||||l|Ill||||||l||||| II | Illlllllllllllllllllllllll Target mRNA gets cleaved and RISC stays bound to target mRNA active RISC is liberated causing translational inhibition Figge 1.5 Target mRNA silencing by active RISC. Active RISC programmed with the guide strand binds with the target mRNA, and cleaves the complementary target. Following the target cleavage active RISC becomes available for more catalytic activity. If the there are mismatches between the guide strand and the target mRNA then RISC stays bound to the target preventing the mRN A translation. RNAi : discovery to application Since its discovery in 1998 the use of RNAi has become a standard procedure for gene regulation. While there is still a lot of progress to be made related to siRNA design and efficacy (Tilesi et al. 2009), stability (Bramsen et al. 2009), controlled delivery (Fattal and Barratt 2009), reducing off-target effects (Jackson et al. 2006), immune response (Judge et al. 2005). Base of design and high specificity of siRNAs make them a convenient tool for, understanding gene functions and regulating their expression. SiRNAs offer novel therapeutic approaches to treat diseases by providing the ability to silence aberrantly expressed genes or to enhance expression of valuable proteins by manipulating cellular pathways (Zhu and Galili 2004; Martin and Caplen 2007). Applications of RNAi in biotechnology and biomedicine Metabolic flux analysis and genetic screens have identified cellular pathways influencing protein synthesis and cell survival (Tewari and Vidal 2003; Srivastava et al. 2007). Those pathways identified as being too active to achieve the desired phenotype can be specifically targeted and their activities reduced using RNAi. One specific application in which this has been demonstrated is in the expression of proteins by mammalian cells, which grow more slowly than bacterial cells, but provide proper folding and post- translational modification of the expressed proteins (Wurm 2004). Since the approval of the tissue plasminogen activator as the first recombinantly expressed therapeutic protein from Chinese Hamster Ovary (CHO) cells, the CH0 cell line and its variations have become the dominant mammalian system for recombinant protein synthesis (Kumar et al. 2008). Maximal protein production from CHO cells 10 requires high viability and initial rapid proliferation of the cells. Apoptosis from stress or other signals reduces the yield and purity of the recombinant protein product. As cells lacking both the proapoptotic proteins BAK and BAX exhibit considerably higher viability compared to cells expressing both these proteins (Wei et al. 2001), siRNAs were used to target these proteins in recombinant human IFN-y expressing CHO cells. With BAK and BAX mRNAs reduced to 10% of their normal level, the CH0 cultures showed 30-50% greater viable cell density, and 35% higher IFN-y production relative to control cells (Lim et a1. 2006). Cellular pathways in plants can also be manipulated using RNAi to engineer and over- express mammalian antibodies and plant specific proteins. Plants have emerged as a safe and economical alternative to microbial and mammalian cell lines for expressing vaccines and therapeutic antibodies (Floss et a1. 2007), in part because they have minimal risk of contamination from potential human pathogens and can be easily scaled up for mass production without the need for extensive purification steps (Daniell et al. 2001). For example, the aquatic plant Lemna minor has been developed for producing high yields of therapeutic recombinant proteins (Neuenschwander et al. 1991). These proteins are, however, susceptible to plant specific glycosylation by the genes a-1,3- fucosyltransferase and B-l,2-xylosyltransferase (Gomord et al. 2005). After silencing these enzymes by vector expressed shRNAs, their activities were reduced to the levels of negative control. When used to produce monoclonal antibodies against human CD30, a human cell surface receptor that is specifically upregulated in certain tumor cells such as Hodgkin and Reed-Stemberg cells, adult T cell leukemia, and embryonal carcinoma of the testis (Dong et al. 2003), the antibodies did not have any detectable plant specific 11 glycans and were more potent than antibodies expressed in mammalian cell lines (Cox et al. 2006). Interestingly, storage organs such as potato tubers can also serve as bioreactors for mass production of human proteins and vaccines, as they provide superb environments for maintaining protein stability (F arran et a1. 2002; Amtzen et al. 2005). A major problem for proteins expressed in tubers is patatin contamination (Amtzen et al. 2005). Patatins are a family of glycoproteins that constitute nearly 40 % of the soluble protein in potato tubers (Prat et al. 1990). shRNA vectors have been used to target the highly conserved gene (pat3-k1) of the patatin family, thereby reducing patatin expression by nearly 99% (Kim et al. 2008). The expressed glycoproteins did not then require purification steps to remove patatin, resulting in significant improvement in protein yield. In cases where the native plant proteins are detrimental, RNAi has been shown to be active in reducing expression of the undesired protein. Peanut allergy is one of the most severe food allergies, affecting nearly 1% of the US population (Sicherer et al. 1999), with 86 % of cases resulting from reactions to the protein Ara h 2 (Koppelman et al. 2004). Knocking down the expression of Ara h 2 using plasmid-expressed shRNAs resulted in hypoallergenic seeds showing significant reduction in allergenic potency, as measured by IgE binding capacity, compared to wild-type peanuts (Dodo et al. 2008). RNAi can also be coupled with metabolic engineering to provide several advantages over classical plant breeding techniques, such as control of spatial and temporal expression of genes of interest (Tang et al. 2007). These new methods of breeding are being investigated to improve the nutritional value of food, offset the loss of agricultural land, and help satisfy the food demand of the growing world population (Doos 2002). For 12 instance, cotton tissues express the protein Gossypol, which acts as a defense against insects and pathogens (Sunilkumar et al. 2006). However, Gossypol's toxicity to humans prevents the use of cotton seeds as a source of food in developing countries (Townsend and Llewellyn 2007). shRNAs targeting the enzyme 8-cadinene, which is crucial for Gossypol synthesis, reduced the Gossypol level in transgenic seeds by 99% compared to wild-type seeds, with the transgenic plant showing normal growth and development (Sunilkumar et al. 2006). A similar approach was used to enhance the plant production of lysine, an essential amino acid important for human nutrition and also livestock growth. Lysine is found in limiting amounts in corn and other cereal grains (Singh et al. 2001), as it negatively regulates the activity of dihydropicolinate synthase (DHPS), the first enzyme in the lysine biosynthesis pathway (Tang et al. 2007). Plants engineered to express DHPS mutants insensitive to lysine showed increased lysine synthesis in all plant organs (F rankard et al. 1992). Unfortunately, elevated lysine levels caused abnormal tissue and flower development, which in turn reduced seed yield (Frankard et al. 1992). The quality of the seeds was also inferior due to defective post-germination lysine catabolism (Zhu and Galili 2004). Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) are key enzymes in the lysine catabolism pathway (Zhu and Galili 2004). In Arabidopsis plants, both the lysine content and seed quality were improved by seed-specific lysine over-expression using both the DHPS mutant and temporal shRNA silencing of the LKR and SDH enzymes during seed development (Zhu and Galili 2004). These seeds had about 25 mol % higher lysine content than wild-type seeds and also grew faster than 13 seeds from plants over-expressing lysine without LKR and SDH knockdown (Zhu and Galili 2004). Alternatively, RNAi modified plants have been shown to be effective for therapeutic drug production. Morphinan alkaloids such as morphine, codeine, oripavine, and thebaine have direct therapeutic use and serve as intermediates for manufacture of synthetic analgesics used to treat opiate addiction (Allen et al. 2008). These alkaloids are only expressed in plants of the genus Papaver (Allen et al. 2008). Upregulating the flux through the alkaloid synthesis pathway by over-expression of the enzymes salutaridinol 7-O- acetyltransferase (SalAT) or codeinone reductase (COR) results in increased production of the alkaloids (Grothe et al. 2001; Allen et al. 2004). Morphine, however, remains a significant product of the pathway, impairing the expression and purification of thebaine and codeine, products of significantly greater therapeutic value. Targeting the COR transcript using shRNAs leads to almost complete inhibition of its enzymatic activity, resulting in the upstream accumulation of (S)-reticuline in the alkaloid biosynthesis pathway (Allen et al. 2004). (S)-reticuline is a valuable pharmaceutical intermediate that can easily be converted to salutaridine and then to thebaine (Page 2005). Engineering poppy plants in this manner is therefore a means to generate plants with significant medicinal value, while also possibly limiting their use in the production of illicit opioid drugs. Summary, aims, and findings of this research The application of RNAi technology to a multitude of platforms frbm bench side to expressing therapeutic proteins highlights it enormous potential. Hence the need to understand and optimize the technology is of critical importance. Several proteins are 14 involved in mediating this endogenous pathway of gene regulation and viral defense (Sontheimer 2005). Dicer and TRBP are two proteins, involved in both the initiation phase of the response and also the formation of the active complex. Similar to siRNAs, 21-nt ssRNAs have also been used as silencing agents in Ag02 mediated silencing experiments (Schwarz et a1. 2002; Rivas et al. 2005). It is not known if Dicer alone can bind 21-nt ssRNAs, as it binds siRNAs (Pellino et al. 2005). PIWI domain of Ag02 has been demonstrated to bind with the RNase 111 domain of Dicer (Tahbaz et al. 2004). Dicer binding with ssRNAs of this length would suggest its ability to interact and load Ag02 with the guide strand, which is also single stranded. Hence the primary aim of this research was to study if Dicer alone could bind with 21-nt ssRNAs. It was found that: l. Dicer alone can bind both 21-nt ssRNAs and siRNAs independent of internal sequence and structure. 2. Only ssRNAs and siRNAs possessing a 5'-phosphate and not 5'-hydroxyl are bound by Dicer, which suggests that this binding is mediated by the Dicer PAZ domain. 3. Dicer binding with ssRNAs and siRNAs is stabilized by different Dicer domains, RNase IIIb domain for ssRNAs and dsRBD for siRNAs. Based on these results a model has been proposed for the Dicer, Ag02 interaction leading to the loading of Ag02 with the single stranded guide strand. 15 Dicer along with TRBP and Ag02, contributes, to the formation of RISC with the siRNA, and selection of the guide strand to form active RISC. To enhance guide strand incorporation into RISC and reduce off target silencing by the siRNA passenger strand, siRNAs are routinely synthesized with a mismatch at the guide strand 5' end (Schwarz et al. 2006; DiFiglia et al. 2007). The effect of such mismatches on the activity of asymmetric siRNAs has not been studied. A comparative study of asymmetric siRNAs having a guide strand 5' end mismatch vs. asymmetric siRNAs without any mismatches, on their interaction with Dicer and TRBP, and also of their silencing activity was performed. The major findings were that: 1. Guide strand 5' end mismatch does not enhance the silencing efficacy of an asymmetric siRNA. 2. SiRNA-TRBP binding is largely indicative of eventual silencing efficacy of the siRNAs and that this binding can be significantly reduced by terminal mismatches. 3. Terminal mismatches led to a small increase in Dicer binding, as expected, however, this did not lead to an improvement in silencing activity. 4. Internal mismatch enhanced both Dicer and TRBP binding. 16 CHAPTER 11 Human Dicer binding of ssRN As and siRNAs INTRODUCTION Dicer is a member of the RNase 111 family of enzymes. There are three classes of RNase III enzymes (Fig. 2.1). Bacterial RNases, with only one RNase domain, comprise class I of the RNase III enzymes. The first RNase 111 family enzyme was isolated from Ecoli and demonstrated divalent cation dependent catalysis of long dsRNAs (Robertson et a1. 1968). These RNases act simultaneously on strands of dsRNAs by dimerization, each monomer possessing an active site cleaves one strand to generate duplexes ~ 10 bp in length. Class II enzymes, such as Drosha, have two endonuclease domains and one dsRBD. Human and Drosophila Dicers are more elaborate and, besides the dsRBD and endonuclease domains, also have the distinguishing PAZ and ATP helicase domains (Zamore 2001). Unlike the bacterial RNase which acts by dimerization, Dicer and Drosha act by intramolecular dimerization of their two endonuclease domains and, assisted by the dsRBD and PAZ domains, cleave the two strands of the substrate generating siRNAs and miRNAs. Though Dicer has been shown to be part of RISC, its exact role in RISC is not well- understood. HeLa cell extracts stripped of Dicer protein by immunodepletion still show siRNA induced target mRNA cleavage activity (Martinez et al. 2002). Also, while Dicer is necessary for normal development of mice embryos (Bernstein et al. 2003; Harfe et al. 2005), mouse embryonic stem cells with an inactive Dicer still retained siRNA directed target mRNA cleavage (Kanellopoulou et al. 2005; Murchison et al. 2005). These results show that Dicer is not a necessary factor for siRNA induced silencing. However, Dicer l7 generated siRNAs from 27-mer dsRNAs have been shown to be better silencers compared to 2l-nt siRNAs with identical base pair composition, possibly due to Dicer mediated incorporation of the siRNAs into the RISC (Kim et al. 2005). Also, Ag02 PIWI domain has been shown to bind directly with the RNase III domain of Dicer (Tahbaz et al. 2004). These results strongly support that Dicer, though not essential for RNAi, can play a role in facilitating siRNA processing and RNAi. This is further supported by in vitro studies that showed that Dicer alone is capable of binding 21-mer siRNAs (Pellino et al. 2005; Kini and Walton 2007). Dicer processing of long dsRNA substrates without mismatches is much more efficient than substrates resembling pre-miRNAs, with internal mismatches (Soifer et al. 2008). The Dicer helicase domain attenuates the processing of substrates with internal mismatches (Ma et al. 2008). In the case of pre-miRNA processing, the helicase domain has been suggested to mediate a conformational change of the enzyme to facilitate product release (Ma et al. 2008; Soifer et al. 2008). Dicer may therefore have the ability to bind with ssRNAs, and be functional in the bypass mechanism suggested in the formation of active RISC programmed with single stranded miRNAs (Matranga et al. 2005) 18 Dicer ATPase/ DUF Helicase 283 PAZ I RNase [Ha I RNase IIIb dsRBD Drosophila Dcr-l ATPase/ DUF ' Heficase 283 l PAZ l l RNase IIIa RNase IIIb dsRBD Drosophila Der-2 ATPase/ DUF Helicase 283 PAZ l RNase IIIa RNase IIIb dsRBD Drosha Pro-rich RS" rich RNase IIIa RNase IIIb dsRBD Bacterial RNase III RNase III dsRBD Figure 2.1 Representation of the various domains of RNase III famin proteins. 19 RESULTS AND DISCUSSION SsRNA-Dicer complex formation in vitro Dicer is known to form stable complexes with dsRNAs and siRNAs (Provost et al. 2002; Zhang et al. 2002; Pellino et al. 2005), though for siRNAs some conflicting reports exist (Provost et al. 2002; Chendrimada et a1. 2005). The focus of this study was to determine if Dicer alone could bind 21-nt ssRNAs. SsRNAs of this particular length were chosen as they are the same length as the individual strands of a siRNA duplex. Also 21-nt ssRNAs have been shown to be functional silencers in Drosophila (Schwarz et al. 2002). For the binding studies, commercially available recombinant human Dicer was used. Silver staining and western blot analysis with monoclonal Dicer antibody for Dicer were done to confirm the size of Dicer protein (~ 210 kDa) and its identity, respectively (Fig. A1, A and B). Dicer functionality was confirmed by cleavage reactions with a 27-mer dsRNA substrate (Fig. A1 C). To study whether Dicer would also form stable complexes with 21-nt ssRNAs, binding reactions were set up with 5'-33P labeled ssRNAs and Dicer (Fig. 2.2A). As a control, complex formation between Dicer and an siRNA was verified (Fig. 2.2A, lane 2). Two bands were seen in the case of siRNA-Dicer binding under identical binding conditions, suggesting the possible presence of ssRNA in the siRNA preparation. Faster migration of the siRNA-Dicer complex relative to the ssRNA-Dicer complex is due to the increase in negative charge on the complex (due to the backbone phosphates of the second strand) with a relatively small change in molecular size/weight (Wu and Regnier 1993). 20 B) siRNA SSl _SSZ_ 27-mer dsRNA Dicer - .1, :1. A f. +- Dicer+ - + + .V‘i g. I C) D) .3 '3 ‘fi 50 c :3 [ii z a z n. i- i f" 3‘“ . E ._ - . <— ‘5 30 O .D E 20 g 10 thcr °' ‘ ' "‘ 33'! 0 500 1000 1500 .' "" , Probability Based Mowse Score 1 2 3 4 F i e 2.2 In vitro bindin of Dicer with siRNA and ssRNAs. (A) Electrophoretic mobility shifi assay of the complex formed between Dicer and, siRNA (lane 2), structured ssRNA SS1 (lane 4), and unstructured ssRNA SSZ (lane 6). The arrow indicates the position of the Dicer-ssRNA complex. Curly brace indicates unbound siRNAs and ssRNAs. (B) Electrophoretic mobility shift assay of Dicer-27-mer dsRNA complex in the presence of Mg + (lane 2) and in the absence of Mg2+ (lane 3). The dotted arrow denotes the Dicer-siRNA complex whereas the solid arrow denotes the Dicer-27-mcr dsRNA complex. (C) Mass spectrometry of the protein-ssRNA complex denoted by the arrow in (A) showed only the presence of Dicer in the complex. (D) Electrophoretic mobility shift assay of Dicer-SS2 complex in the presence of Dicer antibody (Lane 3) and antibody against NF-KB (Lane 4). The asterisk denotes the supershified complex. 21 27-mer dsRNA substrate were carried out. After processing and binding, two bands remained, indicating Dicer complexes with the 27-mer duplex and the 21-mer siRNA product (Fig. 2.2B lane 2). The identities of these bands were confirmed by processing in the absence of divalent cations, which are not required for Dicer binding but are necessary for cleavage (Fig 2.2B, comparing lane 2 and 3) (Zhang et al. 2002). This experiment also provides evidence that Dicer forms stable complex with the siRNAs it generates, and may thereby enhance their incorporation into the RISC complex and also their functionality (Kim et al. 2005; Siolas et al. 2005) . Because Dicer is naturally a dsRNA-binding protein, the secondary structure, or structure formed by intramolecular hybridization, of an ssRNA was expected to impact the ability of Dicer to bind it. So an ssRNA predicted to have secondary structure at 4°C (SS1) and a polyuridine ssRNA (SS2) that is presumably unable to form any significant secondary structure were tested. The reduced electrophoretic motility of unbound SS2 relative to unbound SS1 suggests the general absence of structure in S82 (Fig. 2.2A, compare lanes 3 and 5), supporting the expectations from the TM calculations (Table 2.1). Both SS1 and SS2 are bound stably by Dicer (Fig. 2.2A, lanes 4 and 6), as are other 21-nt ssRNA sequences (Table 2.1). Identical ssRNA-Dicer complexes were also formed with 12 and 15-nt ssRNAs (data not shown). The ssRNA-Dicer complex formed by SS1 with Dicer was analyzed by mass spectrometry to confirm the presence of Dicer as the only protein component (Fig. 2.2C). To confirm further that the complex contained Dicer, an antibody supershift assay was performed. Adding Dicer antibody subsequent to Dicer-RNA binding did not produce 22 Table 2.1 Predicted AG and TM for the sequences predicted using mfold (Zuker 2003). Name Sequence AG, TM: 0C kcal/mole SS1 GUC ACA UUG CCC AAG UCU CT T 0.7 20 SS2 UUU UUU UUU UUU UUU UUU UTT - - SS3 GCU AAA AAA AAA AAA AAA ATT 1.94 -19.3 SS4 GUC AAA AAA AAA AAA AAA ATT 1.94 -19.3 SSS GCU GAC CCU GAA AUU GAU CTT -0.61 32.2 SS6 GAG ACU UGG GCA AUG UGA CUT -1.62 36.7 any shift in the complex, so assays were performed by prior incubation of the antibody with Dicer followed by addition of SS1. This implies that this antibody is a competitive inhibitor of the binding by Dicer of ssRNAs. In the presence of the Dicer antibody, the characterized complex is nearly completely retarded as compared to negative and non- specific antibody controls (Fig. 2.2D, compare lane 3 with lanes 2 and 4). Possible contribution of the PAZ domain to ssRNA binding by Dicer SiRNAs and miRNAs resulting from Dicer processing have characteristic 5'-phosphate and 3' overhangs (Fig 2.3B). Chemically synthesized siRNAs possess a 5'-hydroxyl (Fig 2.3C). Upon entry into cells, these siRNAs are immediately phosphorylated (Fig 2.3B) by the human RNA kinase hClpl (Nykanen et al. 2001; Weitzer and Martinez 2007). SiRNAs with a chemical modification that prevents 5' phosphorylation are bound by Dicer with significantly lower affinity (Pellino et al. 2005; Chen et al. 2008). The presence of a 5' phosphate is an important structural determinant for the guide strand to be incorporated into RISC (Liu et al. 2004). Structural studies of the Ag02 PIWI domain have shown that a conserved metal binding site secures the 5'-phosphate 23 A) ssRNA (SSZ) 5'-0H 5'-P04 ss si ss si 3' PO4-5' C’ 5"0Hjmmirmum—3' 3' OH-5' Figge 2.3 Impact of 5'-phosphate on Dicer binding affinity for ssRNAs and siRNAs. Dicer-ssRNA binding reactions were performed in the presence of 100-fold excess unlabeled ssRNA (lanes 3 and 5) or siRNA (lanes 4 and 6). In lanes 3 and 4, competition was performed with 5'-hydroxyl RNAs. In lanes 5 and 6, competition was performed with 5'-phosphate RNAs. The arrow indicates the position of the Dicer-ssRNA complex. B) Schematic of siRNA structure with 5' —phosphate. C) Schematic of siRNA structure with 5'-hydroxyl. 24 (Parker et al. 2004; Parker et al. 2005; Wang et al. 2008). Anchoring of the 5' end is an important step that determines the position of the target mRNA to be cleaved. Following the formation of this complex, the target mRNA is cleaved between the nucleotides paired to bases 10 and 11 of the guide strand (Elbashir et al. 2001b; Elbashir et al. 2001c). Since ssRNAs have also been shown to induce silencing mediated by the Argonaute protein (Schwarz et al. 2002; Rivas et al. 2005), the impact of the 5'- phosphate on ssRNA binding with Dicer was studied. To assess the impact of 5'-phosphates on complex formation between ssRNAs and Dicer, a competition assay between labeled ssRNAs in the presence of an excess of unlabeled ssRNAs and siRNAs was performed. The competing siRNAs and ssRNAs were chemically synthesized having a 5'-hydroxyl (Fig. 2.3C). At 100-fold excess concentration, neither 5'-hydroxyl ssRNAs nor 5'-hydroxyl siRNAs could displace 5'- phosphate ssRNA SS2 from its complex with Dicer (Fig. 2.3A, lanes 3 and 4). Then, the competing siRNAs and ssRNAs were 5'-phosphorylated using cold ATP (Fig. 2.3B). These phosphorylated siRNAs and ssRNAs effectively displaced bound S82 (Fig. 2.3A, lanes 5 and 6). This suggests a role for Dicer in quality control of the molecules entering the RNAi pathway, by verifying the presence of 5'-phosphate on the silencing agents. Of the domains in Dicer, only the PAZ domain is known to possess a greater affinity for 5'- phosphate ssRNAs as compared to 5'-hydroxyl ssRNAs (Y an et al. 2003). While the 5'- phosphate determines whether the siRNAs and ssRNAs can enter the silencing pathway, the 3'-structure affects the activity of the siRNAs and dsRNAs. DsRNAs with 2-nt 3' overhangs are processed with higher efficiency by Dicer compared to blunt end duplexes, and this interaction is mediated by the Dicer PAZ domain (Zhang et al. 2004). Similarly 25 siRNAs with 3' overhangs have been shown to be better silencers compared to blunt end duplexes (Elbashir et al. 20010). The PAZ domain, containing a conserved oligonucleotide/oligosaccharide binding fold, anchors the 3' overhangs of the duplexes (Song et al. 2003). To explore if the higher affinity for 5'-phosphate substrates seen in ssRNA-Dicer binding was due to the PAZ domain, binding to SS2 with a 5'-phosphate ssRNA and a 3'-biotin was tested. It is known that this modification disturbs PAZ domain-RNA interactions (Y an at al. 2003). It was found that, Dicer has a lower affinity for this sequence relative to 3'-hydroxyl SS2 (Fig. 2.4), further suggesting a role for the PAZ domain in binding to the ssRNAs. 5' terminal nucleotide sequence dependence of Dicer binding The Dicer-ssRNA binding experiments in the presence of competing siRN As showed that the 5'—phosphate is preferred for binding with siRNAs and ssRNAs. Also siRNAs possessing a 5'-methoxy modification at one end of the duplex, which prevents the phosphorylation of that end, leads to the preferential selection of the other strand as the guide strand (Chen et al. 2008). These results indicate that Dicer interacts with the 5' end of both the ssRNAs and siRNAs. Also in Drosophila, it has been shown that during the formation of the RLC, TRBP binds at the 3' end of the guide strand and Dicer 2 binds to the 5' end of the guide strand. Dicer processing of long dsRNAs has been shown to be dependent on the terminal nucleotide sequence (V ermeulen et al. 2005). DsRNA substrates possessing an A at the terminal end were processed much more efficiently than those having a G or C. 26 A) B) ssz SSZ-Q'Bi Dicer - + - + 6 _ L ~ri a .4— s .2 Q 4 - 9 b0 5 .E 2 4 $9 A m -_. ”w °\° m * 0 . I I . 1 2 3 4 532 ssz-3' Bi Fi e 2.4 Dicer has lower affirri for ssRNAs havin a 3'-biotin. (A) Representative figure showing Dicer—ssRNA binding with ssRNA having a 3'- hydroxyl (lane 2) and ssRNA having a 3'-biotin (lane 4). B) Comparison of binding of Dicer to ssRNA having a 3'-biotin to that with a 3'-hydroxyl . Band intensities from lanes 2 and 4 normalized to the corresponding controls in lane 1 and 3 respectively. *, denotes the 2-tailed t-test comparison of Dicer binding to SS2 vs. SS2 -3' Bi for p< 0.05. Therefore, it was tested whether terminal sequence, in addition to terminal structure, was important for Dicer binding to ssRNAs and siRNAs. To look specifically at the influence of the temrinal nucleotides on binding with Dicer, ssRNAs lacking stable secondary structures but with varying terminal nucleotide sequence at the 5' end were designed and tested (Table 2.2). It was observed that Dicer binding to these sequences varied with the terminal nucleotide sequence (Fig. 2.5). 382- AA shows almost two fold higher binding with Dicer than the other sequences. These 27 results indicate that the Dicer interaction with ssRNAs is dependent on the terminal nucleotide sequence. Iable 2.2 Predicted AG and TM for the sequences predicted using mfold (Zuker 2003L Name Sequence AG, TM: 0C kcal/mole SS2 UUUUUUUUUUUUUUUUUUUTT - - SS2-CC CCU UUU UUU UUU UUU UUU UTT SS2-GG GGU UUU UUU UUU UUU UUU UTT 2.5 -17.2 SS2—AA AAU UUU UUU UUU UUU UUU UTT 2.6 -17.9 :1: ' + % Binding to Dicer O r— N w J; U] ON SS2 SSZ-CC SSZ-GG SS2-AA Fi e 2.5 Dicer ssRNA bindin is de endent on the terminal nucleotide sequence. Percentage binding was calculated by normalizing intensity of siRNA-protein complex to the respective unbound ssRNA. *, denotes the 2-tailed t-test comparison of Dicer binding to different ssRNAs vs. SS2 for p< 0.05. 28 Divalent cation dependence of ssRNA-Dicer complex formation While Dicer requires the presence of Mg2+ to be catalytically-active (Zhang et al. 2002), it has been shown to form stable complexes with 100-130 bp dsRNAs even in the absence of Mg2+ (Provost et al. 2002; Zhang et al. 2002). Structural studies with the Aquifex aeolicus RNase III has shown dsRNAs in a non-catalytic complex with the RNase protein, which is structurally different from the catalytic assembly (Blaszczyk et al. 2001; Blaszczyk et al. 2004). The PIWI domain of Ag02 has been shown to interact directly with the RNase domain of Dicer (Tahbaz et al. 2004). These studies show that the Dicer and other RNases can attain alternate structural conformation in a non-catalytic assembly and function primarily as dsRNA binding proteins in the RNAi pathway, and shuttle the silencers to the downstream proteins. In this regard, the effect of divalent cations on the interactions of 21-nt ssRNAs and 21-mer siRNAs with the human Dicer was studied. To test if ssRNA-Dicer complex formation depends on the presence of divalent cations, binding reactions were performed in Mg2+-free buffers. In the absence of Mg2+, Dicer did not stably bind either ssRNA (Fig. 2.6A, lanes 6 and 9, arrow). Complex formation 2+ 2+ 2+ ,2+ 2+ was restored when Mn or Ca were added to the buffer but not Co , N1 , or Zn (Fig. 2.6B, solid arrow). Both in the presence and absence of divalent cations, ssRNAs appear to form smaller complexes (Fig. 2.6B, lane 2 and 8, dotted arrow), but this binding could not be assigned to a specific protein after analysis by mass spectrometry (Fig. A2). Similar divalent cation dependence was not observed for the formation of a stable siRNA-Dicer complex (Fig. 2.6A, lane 3). Comparing lanes 2 and 3 (Fig 2.6A), 29 A) flflii Mg++-++-++- Dicer - + + B) Drcer - + +2 +2+ +2+++ ++ Figge 2.6 Divalent cation dependence of Dicer-ssRNA complex formation. (A) Dicer-ssRNA complexes formed in the presence of Mg2+ (lanes 5 and 8) and not in its absence (lanes 6 and 9). SiRNA-Dicer complex formation was not cation dependent (lanes 2 and 3). (B) Dicer-ssRNA(SS1) complex formation can occur in the presence of + Mg2+,Mn2+, and Ca2+ but not C02+’ Ni2+, or an ' 30 lane 3 does not have the second slower moving complex seen at the top of the gel in lane 2. This is further proof to the presence of ssRNA in the second slower complex as it is . 2+ . . . . . not seen in the absence of Mg .These results indicate that different Drcer domams are involved in forming stable complexes with the siRNAs vs. the ssRNAs. Beyond the recognition of the 5'-phosphate by the Dicer PAZ domains, other Dicer domains may be needed to form stable complexes with either the siRNAs or the ssRNAs. For E. coli RNase III, Mg2+ and Ca2+ act to stabilize complex formation of the enzyme with the bacteriophage T7 R1.1 RNA, which has a hairpin structure (Li and Nicholson 1996). Divalent cations not involved in catalytic activity have been shown to stabilize the interactions between the RNase and the phosphate backbone of the RNA (J i 2006). In the case of siRNAs and other dsRNAs which do not need a divalent cation to bind with Dicer, the dsRBD might play a role. Whereas, in the case of ssRNAs one or both the RNase domains might be needed to form a stable complex. SsRNA binding by Giardia Dicer While the human Dicer is a relatively large (~210 kD), the Giardia Dicer consisting of 756 amino acids is one of the smallest Dicer proteins known (Macrae et al. 2006). It consists of one RNA binding domain, the PAZ domain, and two RNase III domains (Fig. 2.7A). Crystal structure of this enzyme with a dsRNA has been used to demonstrate the details of substrate processing by Dicer. The two metal ion containing RNase 111 domains secure the two scissile phosphates of the dsRNA duplex and cleave the substrate ~25 bp from the end secured by the PAZ domain. It was reasoned that, if the PAZ domain in 31 human Dicer contributes to the binding of ssRNAs, then Giardia Dicer which also has the PAZ domain should also be able to bind ssRNAs. A) l PAZ l lRNase IIIa lRNase [Ilbl B) WM C) 2+ 882 G-Dicer, ng - 250 300 300 Mg + + + + - _ G.Dicer,ng - 150 250 300 300 O O . . Figge 2.7 Schematic of the Giardia Dicer domain and its in vitro binding with ssRNAs. (A) Domains of Giardia Dicer. B) Catalytic activity of Giardia Dicer. Cleavage products of Giardia Dicer enzyme, analyzed by native gel electrophoresis. Arrow indicates the ~25mer siRNA generated (lanes 2 and 3) from cleavage of 35mer dsRNA substrate (lane 1). C) Electrophoretic mobility shift assay of the complex formed between Giardia Dicer and SS2 in the presence (lanes 2-4) and absence of Mg + (lane 5). Arrow indicates the position of the Giardia Dicer-ssRNA complex. Interestingly while the human Dicer can process 25 and 27-mer dsRNA substrates, the G. Dicer cannot process substrates of these lengths (data not shown). Hence the RNase activity of the G. Dicer was tested by the processing of a 35-bp substrate into ~25-mar siRNAs (Fig. 2.7B). Then, in vitro binding reactions with ssRNAs and Giardia Dicer were performed. Similar to human Dicer, Giardia Dicer also binds ssRNAs in Mg2+ 32 dependent manner in vitro (Fig. 2.7C, lanes 2—4). In the presence of EDTA, no stable Dicer-ssRNA complex is formed (Fig. 2.7C, lane 5). Identifying the Dicer domain/s involved in binding with the ssRNA and siRNAs The binding studies showed that human Dicer can bind both siRNAs and ssRNAs. These interactions seemed to involve the PAZ domain and possibly other domains as well. The binding studies with the Giardia Dicer further pointed to the involvement of the PAZ domain. Binding studies in the absence of divalent cations discriminated between binding with siRNAs vs. ssRNAs, indicating the involvement of other Dicer domains in each case. The goal of these studies was to identify the Dicer domain directly in contact with the nucleic acid using photo crosslinking and mass spectrometry. Photo crosslinking has been commonly used to study protein-nucleic acid complexes in diverse cellular processes such as transcription, translation, and DNA replication. UV crosslinking induces the formation of zero-length covalent bonds where protein amino acids and nucleic acid directly contact each other (Shetlar 1980; Bennett et al. 1994). These crosslinked complexes can then be used for structural analysis, characterization of the protein or nucleic acid. Mass spectrometry which provides better sensitivity and specificity compared to traditional methods such as Edman degradation (Merrill et al. 1984; Prasad et al. 1993) has been widely used to determine the protein molecular weight or sequencing the amino acids (Golden et al. 1999; Rieger et al. 2000). A schematic of the experimental setup for the binding site studies is shown in Figure 2.8. After the binding reactions, the samples were UV crosslinked and proteolysed by Trypsin. This series of experiments was designed to identify the peptide fragments 33 Step 1: Dicer-ssRNA binding reaction ssRNA 5'-PO4/V\3'-OH Dicer Step 2: UV cross-linking the Dicer-ssRNA complex & SSRN A UV light 5 ' -PO4 M 3 ' 'OH Dicer Step 3: Proteolysis of UV cross-linked Dicer-ssRNA complex using Trypsin ssRNA 5"PO4 M3'_OH Denotes hypothetical trypsin cleavage d—--u- ‘ -c--q Dicer Step 4: Liquid chromatography-mass spectrometry of proteolysed sample r ------------- F"—~’—_~_ ;----.-------- E Proteolysed , MS _ g S ectra E E sam le System " I g of sample : figure 2.8 Sequence of steps to determine the Dicer domain/s binding with the ssRNA and siRNA 34 which are crosslinked to the ssRNA or the siRNAs. Such nucleic acid bound peptides would have a different mass to charge ratio (m/z), relative to the identical peptide from the Dicer protein alone. Due to the difference in ‘m/z’ the nucleic bound peptide would have a different time of flight than the peptide alone. Overlaying the spectra of the Dicer protein alone with the spectra of either Dicer-siRNA or Dicer-ssRNA would reveal the ‘m/z’ of such peptides. The amino acid sequence of these peptides can then be inferred from the corresponding predicted amino acid sequence (Appendix A, lists details of generating the predicted cleavage map) for the particular ‘m/z’, and thereby identify the - Dicer domain binding to the nucleic acid. Comparison of mass spectrometry generated spectrum peaks with predicted peaks for BSA The experimental setup was tested by proteolysing BSA (Bovine serum albumin) protein using Trypsin. Mass spectrometry data was analyzed using Waters MassLynx software. Multiple charge peaks (m/z) from the BSA peptide fragments were compared to the multiple charge peaks predicted in the cleavage map of BSA for tryptic digest (details in Appendix A) for confirming the tryptic digest of BSA. Figures ABA and A4.A list two of the doubly charged peaks of BSA peptides and the corresponding amino acid sequence (Fig.A3.B and A4.B). Data analysis and predicting the Dicer domain Dicer-ssRNA complex and Dicer Mass spectrometry analysis of the typtic digests of Dicer, Dicer-ssRNA and Dicer-siRNA was then performed. To maintain the same conditions for both the samples the Dicer 35 protein was also UV treated for 10 min prior to trypsinization. Peptide fragments were run through the HPLC column for 75 min and analyzed by the Q-TOF Ultima API system to yield the chromatograms for Dicer protein and Dicer-ssRNA complex (overlaid spectra Fig. 2.9A, individual chromatograms in Appendix A). The spectra from the overlaid chromatograms were analyzed, to identify similar Dicer peptide peaks which are shifted from the fragment peaks of the Dicer-ssRNA complex due to crosslinking to the ssRNA. Fig. 2.9B shows such a peak identified from the overlaid spectra. The spectrum of the Dicer-ssRNA fragments does not have a m/z peak corresponding to the Dicer protein peak of 917.8 (Fig.2.9B, denoted by the arrow). However there are no corresponding peaks, with these m/z predicted by the theoretical cleavage map of the Dicer protein (Fig. 2.9C). This could be due to incomplete cleavage of the protein or due to the presence of the detergent Triton in the Dicer preparation. Cleavage of the Dicer protein was conformed by the presence of several doubly and triply charged peaks indicative of typtic digest. Presence of Triton is the probable cause of variation in the m/z of the peptide fiagments as pure BSA protein digested under identical condition had peptide fragments with the same m/z as predicted by the theoretical BSA cleavage map (Fig. A3 and A4). Detergents like Triton have been shown to interfere with peptide ionization in mass spectrometry (Bomsen et al. 1997; Norris et a1. 2005). Removing the detergent fiom the samples was not a feasible option considering the low yields and microgram amount of protein required to be analyzed by LC followed by mass spectrometry (using Q-TOF). 36 A) 100i ”Ali Time, min 75 B _ 17.8 ) 100 ii 9 918.3 917.4 m % . 918.8 l 919.3 4”,ij o - - . m/z 917 918 919 Figure 2.9 LC/MS chromatoggams A) Overlaid chromatograms of trypsin digested Dicer protein and Dicer-SS complex. B) Spectra of overlaid chromatograms from A) displaying the m/z vales for the Dicer- ssRNA fragments. C) Few predicted peptides resulting from tryptic digest of the Dicer protein, with m/z around 917. 37 C) m/z (mi) m/z (av) Star End Sequence 916.441 917.0122+2 1457 1472 ISLSPFSTTDSAYEWK 917.466 917.0654+3 418 440 EKPETNFPSPFTNILCGIIPVER 917. 843 91 8. 4029+3 1716 1741 OHSPGVLTDLRSALVNNTIFASLA 917.984 918.589fi2 545 560 APISNYIMLADTDKIK figure 2.9 (Continued) Identifying the Dicer domains binding siRNA and ssRNA by comparison of the triple spectra Based on the known roles of RNA domains in Dicer, the PAZ and dsRBD possibly mediated the binding with siRNAs and likewise PAZ- and RNase 111 domains might interact with the ssRNAs. While PAZ domain discriminates between phosphorylated and non-phosphorylated substrates for Dicer binding, it can bind with both siRNAs and ssRNAs having a 5'-phosphate. However different Dicer domain/s are involved in forming a stable complex with either the siRNAs or the ssRNAs. Results from the binding studies support this observation with ssRN As requiring the presence of a divalent cation to bind with Dicer suggesting the involvement of RNase III domains. SiRNAs on the other hand bind with Dicer even in the absence of divalent cations suggesting the RNase 111 domains may not be needed in binding with the siRNAs. Also, the Dicer dsRBD, which has been shown to bind only with a dsRNA and not with ssRNA, RNA- 38 DNA hybrids or dsDNA, might mediate binding with the siRNAs (Bevilacqua and Cech 1996) As unique Dicer domain/s are involved in the binding with the ssRNAs or the siRNAs. Overlaying and comparing the spectra for all the three cases: Dicer protein, Dicer-siRNA complex, and Dicer-ssRNA complex, would differentiate the Dicer domains involved in forming a stable complex with the siRNA or the ssRNA. Also comparing the three spectra with each other gives more confidence in identifying the right peaks by avoiding non-specific variation in the spectral peaks. Figure 2.10A shows the overlaid spectra for the three cases indicating the peaks of the ~ 917 m/z. While both the Dicer protein (Fig. 2.10A) and Dicer-siRNA complex (Fig. 2.108) have a peak corresponding to m/z of 917.8, the Dicer-ssRNA complex does not (Fig. 2.9B). The predicted peak with the m/z of 917.8 has the amino acid sequence of OHSPGVLTDLRSALVNNTIFASLAVK. This sequence corresponds to the Dicer domain RNase-IIIb (Table 2.3). The other predicted peak with m/z value of 917.4 with amino acids from 418-440 (Fig 2.9C), was ignored as it does not correspond to one of the characterized domains of Dicer. 39 A) B) 1 0- - 0 “917.8“ 100 918.3 917.4 % % “918.8 0 . , , 0 917 918 919 m/z 917 918 919 920 m/z Figge 2.10 Overlaid spectra of proteolysed Dicer, Dicer-siRNA and Dicer-ssRNA. The m/z values displayed are for A) the Dicer-siRNA fragments. and B) for the Dicer protein peptides. Table 2.3 Amino acid seguence of human Dicer domains. List of the amino acid sequence for the PAZ, dsRBD, RNase-HIa and RNase-Hlb domains of Dicer. The bold underlined amino acids refer to the sequences indentified by mass spectrometry analyses to be crosslinked with either with the siRNA or the ssRNA. Domain Amino acid sequence PAZ dsstldidfk fmedieksea rigipstkyt ketpfvflde dyqdaviipr ymquphrf yvadvytdlt plskfpspey etfaeyyktk ynldltnlnq plldvdhtss rlnlltprhlnqk (123 aa) dsRBD vprspvrellemepetakfs paertydgkv rvtvewgkg kfkgvgrsyr (66 aa) iaksaaarralrslka RNase-Illa dseqspsigy ssrtlgpnpg lilqaltlsn asdgfnlerl emlgdsflkh (128 aa) aittylfctypdahegrlsy mrskkvsncn lyrlgkkkgl psrrnvvsifd ppvnwlppgy vvngdksntdkwekdemt RNase-IIIb fenfekkiny rflrnkayllq afthasyhyn titdcyque flgdaildyl (159) itkhlyedprghspggltdl rsalvnntif aslavkydyh kyfla-—' mmz _ \.0: ° 1— 0 .. 1 2 C) RNA- DNA- DNA DNA Ext + + Fi e 3.4 Characterizin Dicer TRBP com mlexes in H1299 cell extracts A) Dicer and TRBP complex formation with siRNA in H1299 cell extracts is enhanced by the presence of ATP. B) Quantification of EMSA gel images. Percentage binding was calculated by normalizing the intensities of siRNA-protein complex bands to the respective unbound siRNAs (control lanes not shown). Mean and standard deviation are shown for triplicate binding experiments. C) EMSA of Dicer and TRBP complexes formed in H1299 cell extracts with siRNAs (lane 1), and RNA-DNA heteroduplex (lane 2), and a DNA-DNA duplex (lane 3). Dicer and TRBP complexes form only with the RNA-RNA (siRNA) duplex. 60 396 396-AG 396-UG 396-GG Ext - + - + - + - + . ‘ Sb 1:. 5 ..... > E: r: 8.: ~ "v'- '9‘, 5 l% Binding to TRBP 8 13%Blndlng to cher % Binding to TRBP or Dicer 5‘“ as” at”; e~°° as" 86"“ as“; Figu_re 3.5 Effect of terminal mismatch at gpide strand 5'-end on siRNA-TRBP and siRNA-Dicer complex formation. A) EMSA of siRNA-TRBP and siRNA-Dicer complexes formed in H1299 cell extracts with siRNAs 396 (lane 2), 396-AG (lane 4), 396-UG (lane 6), and 396-GG (lane 8). Separate gels containing other siRNAs not shown. B) Quantification of EMSA gel images. Percentage binding was calculated by normalizing the intensity of siRNA-protein complexes to the siRNA not exposed to extract (e.g., complexes in lane 2 vs. free siRNA in lane 1). Mean and standard deviation are shown for triplicate binding experiments. * denotes the 2-tailed t-test comparison of TRBP binding of different siRNAs vs. siRNA 396 (p < 0.05); 8 denotes the 2-tailed t-test comparison of Dicer binding of different siRNAs vs. siRNA 396 (p < 0.05). 61 Recent work done by our group using purified TRBP protein has shown that it can bind siRNAs in an ATP-independent manner (Gredell JA, Dittrner MJ, and Walton SP, unpublished data). In those studies, TRBP protein by itself did not show a strong preference for binding of firlly matched siRNAs over siRNAs with a terminal mismatch. These results indicate that, in the cells, recognition and binding of the siRNAs by Dicer and TRBP might involve ATP as a cofactor and hence, an in vitro assay using the purified proteins may not capture their behavior completely. That said, both Dicer and TRBP complexes were only formed in the presence of siRNAs and not RNA-DNA hetero duplex or DNA-DNA duplex (Fig. 3.4C), similar to the results with recombinant TRBP protein in vitro. The sensitivity of TRBP binding to the terminal modifications suggests that it primarily binds at the siRNA termini, corroborating its proposed role as a sensor for siRNA asymmetry (Gredell JA, Dittrner MJ, and Walton SP, unpublished data). It has also been shown that an immunopurified complex containing Dicer, TRBP, and Ag02 has the ability to process pre-miRNAs, form active RISC upon selection of a guide strand, and direct Ag02-mediated silencing (Gregory et al. 2005; Maniataki and Mourelatos 2005). Active RISC formed from Dicer processed pre-miRNAs was 10-fold more active than that formed from mature miRNAs targeting the same sequence (Gregory et al. 2005). This is different from the activity of in vitro constituted RLC consisting of only Dicer, TRBP and Ag02 (MacRae et al. 2008). Silencing activity of the RISC formed from the in vitro complex is similar for both pre-miRNAs or miRNAs (MacRae et al. 2008), suggesting in cells there might be other cellular cofactors associated with the RLC and RISC that affect their function. Studying proteins such as MOVIO (Moloney leukemia virus 10 homologue) (Meister et al. 2005; Hock et al. 2007), TNRC6B 62 (trinucleotide repeat containing 63) (Meister et al. 2005), and RHA (DEAH box polypeptide 9) (Hock et al. 2007), which are associated with Ag02 may elucidate the differences between in vitro and in vivo RLC/RISC formation and function. Effect of siRNA structure and composition on siRNA protein complexes The impact of terminal and also selected internal mismatches on the interactions of Dicer and TRBP with siRNAs was fiirther studied (Table B2). G-C rich sequence (used in Fig. 3.3), was used to give the cleanest readout for changes that occurred in the formation of the complexes. A single or double mismatch at one end of the duplex seemed to decrease TRBP binding slightly, though not significantly (Fig. 3.6). For Dicer, binding was improved slightly with a single mismatch, while weakened by the double mismatch. Again, neither of these changes was statistically significant. Simultaneous single or double mismatches at both ends of the duplex significantly reduced binding by TRBP, echoing what was seen with mismatches at only one end. As with one terminal mismatch, binding by Dicer was improved for simultaneous single mismatches but reduced for double mismatches. In all cases, terminal mismatches reduced TRBP binding, as above (Fig. 3.5), strongly suggesting that terminal mismatches should be avoided to generate siRN As with maximal activity. The efficiency of Dicer processing of long dsRNAs is known to depend on the overhang length of the substrates, with 2-3 nt overhangs being highly favorable compared to overhangs longer than 3-nt (V errneulen et al. 2005). In addition, the PAZ (PIWI Argonaute Zwille) domain, which Dicer possesses, is known to mediate binding with dsRNAs and siRNAs through 3'-overhangs (Lingel et al. 2003; Song et al. 2003; Lingel et al. 2004). Binding affinity of the human Ag02 PAZ domain to a siRNA duplex 63 A) 'T ‘7‘ '7 C}! o H N E E E E l l I E ._'. 0'! J. ._'. ..L .L ..L .' ..L .1. .J. CI) U‘ U) (7) CD (0 VJ -—->-—~-—-v a...“- ~ a“ .p....= ..._..-_ . “re-6 1 2 3 4 5 6 7 5 3 I % Binding to TRBP 13% Binding to Dicer * l a N § 96 Binding to TRBP or Dicer O O 1 A a. 6‘ t“ 09"“ F igge 3.6 Effect of terminal and internal mismatches on siRNA-TRBP and siRNA-Dicer complexes. A) EMSA of siRNA-TRBP and siRNA-Dicer complexes formed in H1299 cell extracts with siRNAs of varying terminal and internal structures (Fig. B6). Broken and solid arrows indicate the migration of the siRNA-Dicer and siRNA-TRBP complexes, respectively. B) Quantification of EMSA gel images. Percentage binding was calculated by normalizing intensity of siRNA-protein complex to the respective unbound siRNAs (control lanes not shown). Mean and standard deviation are shown for triplicate binding experiments. * denotes the 2-tailed t-test comparison of TRBP binding of different siRNAs vs. siRNA si-O (p < 0.05); 8 denotes the 2-tailed t—test comparison of Dicer binding of different siRNAs vs. siRNA si-0 (p < 0.05). 64 has been shown to be reduced 5-fold to 50-fold by increasing the overhang length from 2 nt to, 4 nt and 10 nt respectively (Ma et al. 2004). Thus, the assays using cellular extracts seem to accurately demonstrate the natural function of the proteins. Both proteins showed higher affinity for a duplex with one internal mismatch (F ig. 3.6, si-i-mm-l). Binding by TRBP improves with two internal mismatches (Fig. 3.6, si-i-mm- 2), while binding by Dicer is significantly reduced. In relation to Dicer binding, the two internal mismatches are located approximately where the dsRBD (dsRNA binding domain) gets positioned after the PAZ domain binds to one end of the duplex (Lingel et al. 2003; Song et al. 2003), thus the reduction in binding affinity may result from the inability of the dsRBD to bind the disrupted helix (Bevilacqua and Cech 1996) It is possible that the multiple dsRBDs of TRBP assist in its interaction with the sequences with internal mismatches (Chendrimada et al. 2005; Laraki et al. 2008). However, it is not immediately clear why the binding would be improved for the internally mismatched sequence relative to the fully matched control. These structures do resemble miRNAs, and it may be that both Dicer and TRBP have higher affmity for the endogenous silencers as compared to exogenous siRNAs. Also, functional siRNAs tend to have lower internal stability than non-functional siRNAs, particularly at positions 1-6 and 10-15 (with position 1 being the 5'-end of the guide strand) (Khvorova et al. 2003), exactly Where the mismatches are located in this study, which may be due to some still uncharacterized function of TRBP in RNAi. 65 CONCLUSIONS TRBP, Dicer binding of siRNAs and functionality of the siRNAs In this research the interactions of siRNAs possessing terminal mismatches with TRBP and Dicer were characterized, and how these interactions impact their silencing activity. Primarily, it was found that, for an asymmetric siRNA, introducing a terminal mismatch that further reduces the stability of the guide strand 5'-end does not enhance the functionality of the siRNAs. Based on comparison of the binding and silencing results, it is opined that reduced TRBP binding is a likely reason for reduced silencing by mismatched siRNAs. That said, it appears that Dicer binding can impact the silencing efficiency of some siRNAs in a terminal sequence dependent manner. It is interesting to note that all of the mismatches were located at the end where Dicer preferentially should bind based on the current model for RISC formation and siRNA asymmetry sensing (Tomari et al. 2004). Nonetheless, the binding by TRBP is more dramatically and consistently affected. by the mismatches. This assay does not discriminate the location to which either TRBP or Dicer bind on the siRNA. It is possible that TRBP can associate with equal likelihood with either end of the siRNA but that its dissociation rate is faster . with the less-stable end. As such, the mismatches likely enhance this dissociation rate and hence reduce the overall average affinity of TRBP for the mismatched siRNA relative to the fully-paired sequence. Alternatively, this could be a reflection of the importance of the TRBP-Dicer interaction in binding to siRNAs, which would also help to explain the differences between binding with only purified TRBP or Dicer versus binding in extracts. It may also suggest that the role of human Dicer in selecting the guide strand and generating active RISC is more prominent than that of Drosophila Dicer-2, which is 66 controlled by R2D2 binding rather than actively participating in determining which end to bind (Tomari et al. 2004). Future work examining internal and terminal modifications should further identify design rules for enhancing the activity of siRNA duplexes and also provide for a better understanding of the roles of TRBP. and Dicer in controlling siRNA silencing activities. 67 CHAPTER IV Summary and future directions Being an effective and convenient tool to regulate gene expression, RNAi has been widely employed to study gene function and is being pursued as a therapeutic strategy. Hence the understanding of the details of the mechanism of silencing in humans is of great importance to realize its full potential and particularly if it is to be used as a means of therapy. While a great deal of mechanistic details has been learned from the pioneering work done in Drosophila and C. Elegans, there are still several important details yet to be unraveled. Some of these pertain to off-target silencing by siRNAs, the role of the guide strand seed region, terminal stability of siRNAs and selection of the guide strand in RISC, proteins involved in asymmetry sensing in humans, and how the RISC gets programmed with the guide strand in the case of miRNAs without passenger strand cleavage. Dicer and TRBP are the proteins which are primarily responsible for recognizing siRNAs and initiating the formation of RISC. Besides formation of RISC, both proteins are involved in other cellular processes as well. Though they share some known and proposed roles with the corresponding proteins in Drosophila, the human proteins are more complex and might have other functions beyond those that are currently known or can be inferred fi'om their counterparts in other organisms. Major contributions of this research 0 A novel function for both human and Giardia Dicer protein has been found, i.e., ability to bind ssRNAs. 68 0 While both ssRNAs and siRNAs interact with the Dicer PAZ domain, their stable complexes are mediated by different domains: the RNase IIIb domain for ssRNAs and dsRBD for siRNA. 0 Based on the findings of this research and the current understanding of the pathway, a model has been proposed for the interaction between Dicer and Ag02 for loading of Ag02 with the guide strand. 0 The effect of terminal mismatches on TRBP binding has been correlated to the activity of siRN As. Future Work Characterizing other RNAi pathway complexes in cell extracts In vitro silencing experiments to knock down EGFP and GAPDH mRNAs as measured by qPCR have been used to demonstrate the cleavage competency of cell extracts. Complexes formed by siRNAs with Dicer and TRBP proteins in H1299, HepG2 and HeLa cytoplasmic cell extracts have been characterized. It has also been shown that immunopurified complexes containing Dicer, TRBP, and Ag02 have the ability to process pre-miRNAs, form active RISC with the guide strand, and direct Ag02-mediated silencing (Gregory et al. 2005; Maniataki and Mourelatos 2005). Active RISC formed from Dicer-processed pre-miRNAs was lO-fold more active than that formed from identical miRNAs (Gregory et a1. 2005). This is different from the activity of in vitro constituted RISC consisting of only Dicer, TRBP and Ag02 (MacRae et al. 2008). Silencing activity of this RISC is similar for both pre-miRNAs or miRNAs (MacRae et al. 2008), suggesting in cells there might be other cellular cofactors associated with the RISC proteins that affect its function. Studying proteins such as MOVIO (Moloney 69 leukemia virus 10 homologue) (Meister et a1. 2005; Hock et al. 2007), TNRC6B (trinucleotide repeat containing 6B) (Meister et al. 2005) , and RHA (DEAH box polypeptide 9) (Hock et al. 2007), which are associated with Ag02, may elucidate the differences between in vitro and in vivo RISC function. Other sequence and structural modifications could be used to study their effect on interactions with the proteins of the RNAi pathway as the siRNA progresses from a duplex to single stranded guide strand. Is it RNAi ? Results fi'om this research have shown that both TRBP and Dicer can bind only to RNA- RNA duplex, and not to a RNA-DNA or DNA-DNA duplex. (Fig. 3.4C). These results are supported by earlier reports demonstrating the necessity of A-form of helix formed by a RNA-RNA duplex in the siRNAs and between guide RNA and mRNA for effective silencing (Chin and Rana 2002). Furthermore, A-form helix formed by the guide strand and mRNA is necessary for endonucleolytic cleavage by Ag02 (Parker et al. 2004; Parker et al. 2005). However there are reports with duplexes with DNA substitutions in the sense and antisense sequence inducing moderate to active silencing, comparable to siRNAs with the same sequence (Hohjoh 2002; Ui-Tei et al. 2008). It is possible that there are other proteins and mechanisms different from RNAi mediating the silencing in these cases. The in vitro cell extract system can be used to identify the protein complexes formed by these modified duplexes to understand the mode of silencing. This strategy can also be employed to study silencing by siRNAs with modifications which alter the basic structure of the duplex, 19 base pairs, 5'-phosphate and 3' overhangs. Such shorter siRNAs have been tested and found to be effective silencers (Hohjoh 2004; Chang et al. 2009). 70 siRN As with terminal and internal modifications Among all the sequences with a terminal mismatch only 396-AG exhibited silencing efficacy comparable to 396 (Fig. 3.1). It appears that Dicer binding does impact the silencing efficiency of siRNAs in a terminal sequence dependent manner. While the terminal mismatches seem to have an adverse impact on TRBP, one internal mismatch seems to enhance both the TRBP and Dicer binding (Fig. 3.6). Future work with siRNAs having internal and terminal modifications should identify design rules for enhancing the activity of siRNA duplexes and also provide for a better understanding of the role of TRBP and Dicer in controlling siRNA silencing activity. Identifying siRNAs that are functional in the absence of TRBP might be useful for targeting genes in cell types with low TRBP expression, such as astrocytes (Gatignol et a1. 2005; Christensen et al. 2007). Binding studies with Dicer mutants The Dicer helicase domain has been observed to regulate the processing of pre-miRNA like substrates and not dsRNA substrates (Ma et a1. 2008; Soifer et al. 2008). So the structural rearrangement of the substrate protein complex must be different for a Dicer- miRNA complex vs. a Dicer-siRNA complex. It would be interesting to know if mutations in the helicase domain that enhance pre-miRNA processing would also affect the ability of Dicer to bind with ssRNAs. This would provide further evidence to the involvement of Dicer in the bypass mechanism of Ag02 loading. 71 APPENDIX A Characterizing Dicer protein preparation A) B) C) M Dicer D' Icer 27-mer dsRNA + + i E Dicer M + - Figge A1 Characterizing ssRNA-protein complexes Dicer protein visualized by A) Silver staining B) Western blot with monoclonal Dicer antibody. C) Dicer cleavage of a 27-mer dsRNA visualized by native gel electrophoresis 72 Mass spectrometry analysis of unknown complexes formed by ssRNAs Number of Hits l * l .-—r'. . . 1000 Probability Based Mowse Score Figure A2 Mass spectromemy of the fa_ster moving complexes formed by the saRNA (denoted by the dotted arrows in Figu_re 2.6). Proteins with sigpificant hits identified bv mass spectroscopyI keratin 1 (Homo sapiens), keratin 10 (Homo sapiens), keratin 9 (Homo sapiens), Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-Ze) (K26), cytokeratin 8, keratin 5 (Homo sapiens], keratin K7, type II, epithelial, 55K - human keratin, 65K type II cytoskeletal - human, TPA: Homerin (Homo sapiens), NuMA protein (Homo sapiens), alpha 1A adrenoceptor isoforrn 2b (Homo sapiens), KIAA1481 protein (Homo sapiens), putative (Homo sapiens) 73 Generating theoretical cleavage map for tryptic digest of a protein Mass spectrometry data analysis: BSA The cleavage map were generated from the University of California, San Fransisco ProteinProspector website : 'http://prog)cctor.ucsf.cdu/cgi-bin/msform.cgi?form=msdigcst Following options were chosen for tryptic digest of BSA (accession number: P02769): Database: SwissProt Digest: Trypsin Missed clevages: 1 End modification: carbamidomethyl Peptide mass range: 250-4000 Multiple charges to be displayed 74 A) 1 00- 499.3 499.8 °\° _ l i if ' 500.3 590.4 ' 500.9 497.3 5°24 0.. h 1' . i I ': __-9.Q ,,4_92__.49_4_ 496 198 500 -502 50.5 5262-50.11 B) m/z (mi) m/z (av) Start End Sequence 496.6363+3 496.9465+3 549 561 (K)QTALVELLKHKPK( 499.3001” 499.6112"2 549 557 (K)QTALVELLK(H) 501.2982+2 501.6096+2 233 241 (R)ALKAWSVAR(L) 502.3118+3 502.6234” 549 561 (K) QTALVELLKHPK(A) F_igure A3 Spectra of trypsinized BSA protein A) A doubly charged peak of BSA peptide, and B) the corresponding peptide sequence in bold. 75 784.4 ' A) 103. ' 7/84.9 785.4 | l ! 785.9 1‘ ii iii 786.4 I O I .. -2_ Ahancha'xw M-M~-‘su U is"; in}; :Lj‘lfi. A 7 - B) T 776 I 778 r 780 j 782 I 784 I 786 788 m/z (mi) m/z (av) Start End Sequence +2 +2 347 359 784.3750 784.8721 (K)DAFLGSFLYEYSR(R) 785.718 1+3 784. 2 1 68+3 402 420 (K)HLVDEPQNLIKQNC(L) 788.8874” 789.4074” 139 151 (K)LKPDPNTLC(A) 789.4716 789.9529 257 263 (K) LVTDLTKV) Figure A4 Spectra of trypsinized BSA protein A) A doubly charged peak of BSA peptide, and B) the corresponding peptide sequence in bold. 76 A) 100 “ W 04 \j \ 2 C l B) 100‘ i l “A % . ll . l l i “ gill)! ," v ll Ii ‘2 If \MJKJ‘L‘x/M U _..-..- . -._»/ ‘VLVK-M‘ ...__ 0 a- .. W'Time,min . 75 C>100 Figure A5 LC/MS chromatogiams Chromatograms of A) proteolysed Dicer B) Dicer- siRNA complex and C) overlaid chromatograms from A and B. 77 Methods and materials RNA and 5’-end labeling HPLC purified RNAs were purchased from Invitrogen (Carlsbad, CA) or Dharrnacon (Lafayette, CO). Lyophilized RNAs were resuspended in 100 mM concentration Tris- EDTA (TE, pH 8.0) and stored at -80°C. RNAs were 5'-end labeled with 33P-y—ATP (Perkin Elmer Life and Analytical Sciences, Boston, MA) using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). Labeled strands were purified from unincorporated label by G-25 Sephadex columns (Roche Applied Science, Indianapolis, IN). For nonisotopic labeling, unlabeled ATP (Roche Applied Science, Indianapolis, IN) 33P was substituted for -y-ATP. The ssRNA and siRNA sequences used were: siRNA, 5'— GCUGACCCUGAAGUUCAUCUU-3' (Sense strand), 5'-GAUGAACUUCAGGGUCA GCUU-3' (Anti-sense strand); ssRNA-SS1 5' —GUCACAUUGCCCAAGUCUCTT-3'; ssRNA- SS2, 5'- UUUUUUUUUUUUUUUUUUUTT-3'. SsRNA with the 3'-biotin had the same sequence as SS2 but with a 3'-biotin Characterization of recombinant human Dicer purity Recombinant human Dicer (Invitrogen) was used in all the binding reactions. Full length Dicer protein was visualized by Silver Staining Kit (Bio-Rad) (Figure AlA) and confirmed by Western blot (Figure AlB). For western blot analysis, proteins were initially electrophoresed at 150 V for 1.5 hours at 4°C on 4-20 % TBE gels (Bio-Rad) and then transferred to nitrocellulose membranes for 1h at 100V, followed by incubation with mouse monoclonal antibody for Dicer overnight at 4°C. Blots were washed and then 78 incubated with HRP-linked secondary antibodies (Pierce Biotechnology, IL, USA) for 1 h. After an additional wash, the blots were developed with Pierce SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and imaged on Chemidoc XRS imager (Bio-Rad). Dicer activity was assessed through a cleavage assay using a 27- mer dsRNA substrate being converted to ~ 21-mer siRNAs (Figure AlC, compare Lanes 2 and 3). Dicer-RNA binding assays Dicer binding assays were carried out in 30 mM Tris-HCl (pH=8.0), 250 mM NaCl, 2.5 mM MgC12, and 0.02 mM EDTA. Labeled RNA, and 0.5 U of Dicer (Invitrogen) were incubated at 4°C for 2 hours in 10 ul reaction volumes. For antibody supershift assays, either Rabbit polyclonal antibody to Dicer (Abcam) or control antibody was incubated with Dicer at 4°C for 3 hours after which the labeled RNA was added and incubated for 2 additional hours. Samples were electrophoresed at 150 V for 1.5 hours at 4°C on 4-20 % TBE gels (Bio-Rad). Gels were dried under vacuum at 80°C, exposed to storage phosphor screens, and imaged on a Storm 860 imager (GE Healthcare/Amersham Biosciences, Piscataway, NJ). Binding reactions to test the divalent cation dependence were canied out with the appropriate divalent cation substituting for Mg2+ in the binding buffer. Mass spectrometry was performed on gel purified protein by the Michigan State University Research Technology Support facility. 79 In-solution trypsinization 1. Protein was dissolved in 50 mM ammonium bicarbonate of Tris buffer (pH 8) at a concentration of approximately 1-10 uM. Dithiothreitol (DTT), tris-carboxyethylphosphine (TCEP), or tributylphosphine was added to 10 mM final concentration and heated at 95 0C for 10 minutes and cooled to room temperature. Freshly made iodoacetamide was added to 200 uM final concentration and incubated for 1 hour at room temperature preferably in a dark drawer or wrapped in aluminum foil. DTT was added to 1 mM to quench the ioodacetamide. 5. After 10 mins, 30 ng of freshly diluted trypsin (Sequencing grade, Promega) prepared in 50 mM ammonium bicarbonate, pH 8 was added, and incubated at 37 0C for 4-24 hours. An equal volume of 5% Formic acid was added and sonicated in water bath for 10 minutes to quench the digestion, and redissolved in a minimal volume of water/acetonitrile (90:10 v/v) containing 1% formic acid for LC/MS analysis. LC/MS Hypurity Aquastar Pioneer (Thermo Fisher Scientific) column was used in the Waters 2795 separation module. The mobile phase had a mixture of 0.1 % Formic acid (A) in water and acetonitrile (B). The following gradient was used during the run : Time, min 0 2 40 45 55 70 70.01 75 B 1 1 85 85 99 99 l 1 A 99 99 15 15 l 1 99 99 Flow through fi'om the column was then passed through Q-TOF Ultima API) system (Waters) under positive electron spray having a cone and capillary voltage of 35 V and 3 KV respectively. 80 APPENDIX B l2nM IlsnM EminM .20 nM I40nM Normalized RFU y? %% \\. //// /’_ Fi e B1 EGFP silencin efficac of siRNAs at different concentrations EGFP-expressing H1299 cells were transfected with siRNAs targeting the EGFP mRNA or a non targeting (NT) siRNA at final concentrations of 2, 5, 10, 20 or 40 nM. Fluorescence was measured 24 hours after transfection. Mean and standard deviation are shown for 12 wells for each condition. Control, mock, and NT refer to untreated cells, cells treated with the transfection reagent alone, and cells transfected with the NT siRNA, respectively. 81 A) NT TRBP B) NT Dicer Control siRNA siRNA Control siRNA siRNA TRBP Dicer ab ab B-actin ,, . ab m4 ,7 s. .2 an B-actrn - IIII - ab .. 1 2 3 1 2 3 C) E 7 _ - 3, 1'“ DTRBP 8 1 _ IDicer I- St 0 E, 0.8 w , p. O o as .2 as. 0.6 -— 1 — a o h H :1 g 0.4 -1 ‘5 E 7, 0.2 - l- 0 1 . Control NT TRBP/Dicer Fi e BZ Western blot anal sis of TRBP and Dicer levels in H1299 cells. A) Comparison of TRBP knockdown (lane 3) relative to control (lane 1) and transfection with a non-targeting siRNA (lane 2). B) Comparison of Dicer knockdown (lane 3) relative to control (lane 1) and transfection with a non-targeting siRNA (lane 2). C) Band intensities were quantified using Bio-Rad Quantity One software. Quantification of TRBP or Dicer expression relative to B-actin; control ratio normalized to l. * denotes the 2-tailed t-test comparison of TRBP or Dicer expression relative to control (p < 0.05). Mean and standard deviation are shown for triplicate silencing experiments. 82 A) Q C‘fi $00$f§ «‘6’ “no Cir-~69 W“ ' " ' ‘— ‘1' n , . 1‘ l - 12 3 4 B) 5 4 T O. E ,_3 3 £2 E ... m 'r 331 ' 0 fl , j CM Hock NT TRBP Figure B3 Characterization of siRNA-TRBP complex formation after TRBP knockdown A) EMSA of siRNA-TRBP complex formed in H1299 cell extracts with siRNAs. Control, mock, NT, and TRBP refer to extracts fiom untreated cells, cells treated with the transfection reagent alone, cells transfected with the NT siRNA, and cells transfected with the TRBP-targeting siRNA, respectively. Arrow denotes the position of siRNA- TRBP complex. B) Quantification of gel images. Percentage binding was calculated by normalizing the intensity of siRNA-protein complexes to the siRNA not exposed to extract (e.g., complexes in Lane 2 vs. unbound siRNA (not shown)). Mean and standard deviation are shown for triplicate binding experiments. 83 B) % Binding to TRBP 0 __—_ 306 306-CC Fi B4 Effect of a terminal mismatch at the uide strand 5’ end on siRNA-TRBP complex formation. A) EMSA of siRNA-TRBP formed in H1299 cell extracts with siRNAs 306 (lanes 1 & 2) and 306-CC (lanes 3 & 4). B) Quantification of EMSA gel images. Percentage binding was calculated by nomalizing the intensity of the siRNA-TRBP complex (lanes 2 and 4) to the respective unbound siRNAs (lanes 1 and 3). Mean and standard deviation are shown for triplicate binding experiments. * denotes the 2-tailed t-test comparison of TRBP binding of siRNA 306-CC vs. 306 (p < 0.05). 84 A) 13) G‘ J. Fi e B5 Effect of a terminal mismatch at the uide strand 5' end on siRNA-TRBP complex formation. A) EMSA of siRNA-TRBP complexes formed in H1299 cell extracts with siRNAs 274 (lanes 1 & 2) and 274-UG (lanes 3 & 4). B) Quantification of EMSA gel images. Percentage binding was calculated by normalizing intensity of siRNA-TRBP complex (lanes 2 and 4) to the respective unbound siRNAs (lanes 1 and 3). Mean and standard deviation are shown for triplicate binding experiments. * denotes the 2-tai1ed t-test comparison of TRBP binding of siRNA 274-UG vs. 274 (p < 0.05). 85 | si-mm-O —(i> "l' \'T $“f"?"?“?“f‘?“? .“?“‘f"?*?‘fi"?“?*?*fi’ .;-—0—0—o—o—o—0—o—o-o—Q—o—o—o—0—0—o—0—0—Q -;|> si-mm-l ° : (3’61”?—‘9’?’?’?’?‘?_?_?‘?—?_?_?_?—?_?“T( o—o-o—o—o—m—o—o—o—T—o-o-o—o—o—o—a—T an I w 03 ‘ | in c» IIIIIIIIIIIIIIII ,,J-\ /O—0—0—0—0—0—O—(D—(9—0—O—O-0—O—0—0 \ \<_ si-mm-2 (L 'T my. si-i-mm-2 o (I) 0 I— (L—o—o—o—co/ \o—o— —o—o—o—0/ \o—o—o—o— I I I I l I I l l l l | l l I I l /1-~0—0—0—0—0\0/0—0—0—0—0—0—0 0—0—0—0— \o/ —C—G—:l' F_igure B6 (Continued) Terminal stability analysis of siRNAs with terminal mismatches Iable B1 SiRNAs with terminal modifications (Dirg et al. 2007) AAG, Name kcal/mol Antisense sequence (5' - 3') Sense sequence (5' - 3') GAGACUUGGGCAAUGUGA GUCACAUUGCCCAAGUCUC P11 0.1 CT 'I'T GAGACUUGGGCAAUGUGA GUCACAUUGCCCAAGUCUA A1 2.2 CT TT 87 Iable B2 Sequence of siRNAs used to ta_rget EGFP Name Antisense sequence(5' - 3') Sense sequence (5' - 3') si-396 CAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUGTT si-396-AG AAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUGTT si-396-UG UAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUGTT Si-396-GG GAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUGTT si-396-CA CAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUATT Si-396-CU CAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUUTT si-396-CC CAGGAUGUUGCCGUCCUCCTT GGAGGACGGCAACAUCCUCTT Si-306 CUUGUAGUUGCCGUCGUCCTT GGACGACGGCAACUACAAGTT Si-306-CC CUUGUAGUUGCCGUCGUCCTT GGACGACGGCAACUACAAATT Si-274 UGCGCUCCUGGACGUAGCCTT GGCUACGUCCAGGAGCGCATT si-274-UG UGCGCUCCUGGACGUAGCCTT GGCUACGUCCAGGAGCGCGTT Dicer- UUUGUUGCGAGGCUGAUUCTI‘ GAAUCAGCCUCGCAACAAATT siRN A "1:11:11: GCUGCCUAGUAUAGAGCAATT UUGCUCUAUACUAGGCAGCTT N: or si-O CCGGGCCGGCCGGCCGGCCTT GGCCGGCCGGCCGGCCCGGTT si-mm-l ACGGGCCGGCCGGCCGGCCTT GGCCGGCCGGCCGGCCCGGTT Si-mm-2 AAGGGCCGGCCGGCCGGCCTT GGCCGGCCGGCCGGCCCGGTT Si-l-mm-l ACGGGCCGGCCGGCCGGCGTT AGCCGGCCGGCCGGCCCGGTT si-2-mm-2 AAGGGCCGGCCGGCCGGGGTT AACCGGCCGGCCGGCCCGGTT si-i-mm-l CCGGGCCGGGCGGCCGGCCTT GGCCGGCCGGCCGGCCCGGTT si-i-mm-Z CCGGGGCGGCCGGGCGGCCTT GGCCGGCCGGCCGGCCCGGTT RNA-RNA GGCUACGUCCAGGAGCGCAUU UGCGCUCCUGGACGUAGCCUU RNA-DNA GGCUACGUCCAGGAGCGCAUU TGCGCTCCTGGACGTAGCCTT DNA-DNA GGCTACGTCCAGGAGCGCATT TGCGCTCCTGGACGTAGCCTT 88 Iable B3 siRNAs with terminIal modifications (Holen et al. 2005) % AAG, Name kcal/mol Silencing Antisense sequence (5' - 3') Sense sequence (5' - 3') ~70 GCACUCAUCAUUGUGCUGCU F6775 1.3 GCAGCACAAUGAUGAGUGCAA U ~70 ACACUCAUCAUUGUGCUGCU mlAaS 4-7 GCAGCACAAUGAUGAGUGCAA U ~70 CCACUCAUCAUUGUGCUGCU mICaS 4-7 GCAGCACAAUGAUGAGUGCAA U ~70 UCACUCAUCAUUGUGCUGCU mans 47 GCAGCACAAUGAUGAGUGCAA U Calculating terminal stability of the siRN As Terminal stability (AG, kcal/mol) at each end of the siRNA duplex was calculated using Mfold (Mathews et a1. 1999; Zuker 2003) by summing up the base pairing energy of the initial four base pairs at that 5' end (Schwarz et al. 2003) (Fig. B7). Differential end stability (AAG, kcal/mol) was calculated by taking the difference in thermodynamic stabilities at each end. Sample calculations for differential end stability of a siRNA duplex : 5'- ACGCUGAACUUGUGGCCGUTT - 3' Antisense strand(AS) 5'- ACGGCCACAAGUUCAGCGUTT — 3' Sense strand (SS) Step 1: Go the URL mentioned below, http://dinameltbioinforpi.edu/results/twostate/08l201/15103l/ 89 Step 2: In the lefi box paste the AS sequence from 5' to 3' In the right box paste the SS sequence from 5' to 3' and submit it to the Mfold server. Step 3: After Mfold hybridizes the sequences it leads to the page with TM, siRNA structure, Thermodynamic details etc. Click on the Thermodynamic details. Sum the AG for the rows 2-5 of the table (Fig. BSA) to get the terminal stability of the antisense end AGAS, kcal/mol. (fl. .5 O :. . art-rfi-rrrrrmrc-rn‘. {c-o-oic-ro—Jv—ro— o-o->-o-:o—>-o—_ii' -( 6 O —-l F_igure B7 Structure of siRNA. The dotted box shows bases used in calculating stability at that end of the duplex Step 4: Repeat Steps 2 through 3 by pasting the SS sequence in the left box and the AS sequence in the right box for Step 2 to calculate the terminal stability of the other end of the siRNA, AGSS, kcal/mol (Fig. B8.B). Step 6: Differential end stability, AAG = AG AS - AGSS. =-9.2 - (- 10.4) =-1.2 kcal/mol 9O A) Structural element AG Information External loop -O.1 2 ss bases & 1 closing helices . . . l 19 Stack -2.1 External closmg pair IS A -U . . . 2 18 Stack .20 External closmg pair is C -G . . . 3 I7 Stack .29 External closmg pair 18 G -C . . . 4 l6 Stack -3.4 External closrng pair is G -G B) Structural element AG Information External loop -0.1 2 ss bases & 1 closing helices . . . I 19 Stack -2.1 External closrng pair 18 A -U . . . 2 18 Stack .20 External closmg pair 18 C -G . . . 3 l7 Stack -3.4 External closmg pair 18 G -C . . . 4 l6 Stack -1.7 External closmg pair 18 C -G Figure B8 Mfold 2 state hybridization server webpage listing thermodypamic detail; Tables show contribution and base pairing energy of first four base pairs at A) Antisense strand end and B) Sense strand end respectively. 91 Methods and materials General methods siRNAs were purchased from Thermo Scientific Dharmacon (Lafayette, CO). Lyophilized RNAs were resuspended to 100 uM in TE (pH 8.0) and stored at -800C. RNAs were 5'-labeled with 33P-y-ATP (Perkin—Elmer Life and Analytical Sciences, Boston, MA) using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). Labeled strands were purified from unincorporated label using G-25 Sephadex columns (Roche Applied Science, Indianapolis, IN). Cell cytoplasmic extracts were prepared as described (Lee et al. 1995). Binding reactions in cell extracts with radiolabeled siRNAs were performed as described (Pellino et al. 2005). All binding reactions are performed for l h at 37°C. The competency of all extracts for in vitro silencing was tested by measuring EGFP mRNA transcript levels in H1299 cell cytoplasmic extracts prior to and following addition of siRNAs (data not shown). EMSAs was performed as previously described (Kini and Walton 2007) and quantified using a Storm 860 imager (Amersham/GE Healthcare, Piscataway, NJ). Percent binding was calculated by normalizing the intensity of the siRNA-protein complex (Fig. 3.5A, lanes 2, 4, 6 and 8, complexes indicated by arrows) to that of the respective unbound siRNA (Fig. 3.5A, lanes 1, 3, 5 and 7). Sequences of all RNAs used in these studies are listed in Table B2. ATP depletion experiments were carried out in binding buffer lacking ATP and containing glucose and hexokinase without creatine phosphate and creatine kinase (Pellino et al. 2005). 92 Cell transfection and EGFP quantification Human lung carcinoma cells (H1299) constitutively expressing EGFP were generously provided by Dr. Jorgen Kjems, Department of Molecular Biology, University of Aarhus, Denmark. They were maintained in Dulbecco’s modified Eagle’s medium complemented with 10 % (vol/vol) fetal bovine serum (Invitrogen), 100 mg/ml of penicillin, and 100 units/ml streptomycin (Invitrogen). 24 h before transfection, cells were seeded at 50,000 cells/well in 24-well plates in antibiotic free media for siRNA transfection or seeded at 400,000 cells/well in 6—well plates for TRBP plasmid DNA transfection. Cells were transfected using Lipofectamine 2000 (Invitrogen) (0.8 uL for siRNA transfection and 3 uL for plasmid transfection), according to the manufacturer’s recommendations. SiRNA or TRBP plasmid DNA was diluted with Opti-MEM (Invitrogen) followed by addition of Lipofectamine and complex formation. SiRNAs were used at final concentrations of 10 nM and TRBP plasmid DNA at 1 pg. When two siRNAs were transfected simultaneously, the final, total siRNA concentration was 20 nM. Cells were treated with this transfection medium for 4 h at 37°C after which the transfection medium was replaced with regular cell culture medium. 24 h afler transfection, the culture medium was aspirated and EGFP levels were quantified as described (Gredell et al. 2008). It was previously confirmed that transfection efficiency using established protocols provides essentially uniform siRNA loading across the different siRNA treatments (Gredell et al. 2008). For EGFP quantification, the fluorescence of each well of the 24-well plates was measured in 9 locations using a Gemini fluorescence plate reader (Molecular Devices, Sunnyvale, CA). The mean fluorescence for each well was calculated from these 9 93 values. The average fluorescence for a condition was calculated as the mean of multiple wells (typically 3-4) on the same plate. Relative fluorescence units (RFU) (Fig. 3.1 and 3.2) were calculated by normalizing the multi-well average fluorescence for each condition to the multi-well average fluorescence of mock transfected wells from the same plate. At least three wells from at least six different 24-well plates were measured for each condition (11 Z 18). Western Blots Cells were collected 24 h after plasmid or siRNA transfection. SDS-loading buffer was added to samples and heat denatured at 950C for 5 minutes. The samples were immediately placed on ice, and the proteins were resolved on 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad) at 150V for 90 minutes. Proteins were then transferred to a PVDF membrane at 100V for l h. The membrane was then incubated with blotting grade milk (Bio-Rad) for 1 h and then incubated overnight at 4°C with either TRBP antibody (Abnova, Walnut, Ca) or Dicer antibody (Abcam, Cambridge, MA) at 1:1000 dilution. Blots were then washed with TBS-Tween and incubated with the horseradish peroxidase-conjugated secondary antibody, and the proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, IL). B-Actin was used as the loading control. Dicer and TRBP knockdown levels were measured by normalizing to the control cells (no transfection). Images were collected using a ChemiDoc XRS (Bio-Rad, Hercules, CA), and band intensities were quantified using Bio-Rad Quantity One software. Dicer and TRBP knockdown were quantified by a ratio of ratios. Dicer and TRBP levels were normalized to B-Actin loading control for 94 each treatment, and these ratios were then normalized to the ratio for control cells (no transfection). Free Energy Calculations Terminal stability (AG, kcal/mol) at each end of the siRNA duplex was calculated using mfold (Mathews et al. 1999; Zuker 2003) by summing the nearest neighbor contributions for the first five nucleotides (four nearest neighbor energies) at the 5' end (as in (Schwarz et al. 2003)). Differential end stability (AAG, kcal/mol) was calculated by taking the difference in thermodynamic stabilities at each end. For example, si-396 has guide strand sequence of 5'-CAGGAUGUUGCCGUCCUCCTT-3' and passenger strand sequence of 5'-GGAGGACGGCAACAUCCUGTT-3'. Base pairing energies for the duplex were predicted using the mfold 2-state hybridization server with RNA selected and default parameters. The four nearest neighbors at the guide strand 5' end, CAzGU, AGzUC, GG:CC, and GA:CU, have a cumulative base pairing energy of -8.7 kcal/mol. The four nearest neighbors at the passenger strand 5' end, GG:CC, GA:CU, AGzUC, and GG:CC, have a cumulative base pairing energy of -9.8 kcal/mol. Consequently the differential end stability, i.e., the thermodynamic asymmetry, for the duplex is AAG = 1.1 kcal/mol. Positive values of AAG indicate that the sequence is asymmetric in favor of the appropriate guide strand. qPCR Total RNA was extracted using RNeasy Plus Mini Total RNA Purification Kit (QIAGEN) according to manufacturer’s instructions. cDNA was synthesized using iScript Select cDNA Synthesis Kit (BIO-RAD laboratories) with a final volume of 20 uL 95 including 4 uL of 5X reaction mix, 1 uL of reverse transcriptase and 1 ug of RNA in every reaction tube. Quantitative real time PCR was performed using iQTM SYBR® Green Supermix (BIO-RAD laboratories) on the synthesized cDNA with 12.5 uL of iQ SYBR green super mix, 5 uL primers(lOOnM) of GFP ( forward CACCTACGGCAAGCTGACCCTGAA, reverse CCCTTCAGCTCGATGCGGTTCAC) and the , 0.5 uL of water and 2.0 uL of cDNA with each biological sample having three replicates. 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