Structural biology on RNA silencing suppressors and their potential targets

Structural Biology on RNA Silencing Suppressors and Their Potential Targets YANG JING (Master of medicine, Beijing Univ of Chinese Medicine and Pharmacology, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009     II    To my family   III    Acknowledgement I would like to express my deepest gratitude to my supervisor, Dr. Adam.Yuan, for his invaluable guidance, advice and mentorship. Thanks for giving me an opportunity to commence my research work in his lab and providing a motivating, enthusiastic, and critical atmosphere for my work. I am deeply indebted to Ms. Chen Hongying, for her contribution to TAV2b structure determination. I also greatly thank for her selfless assistance and support in the technical guidance of my research projects. I would like to thank for Mr. Lin Chengqi, for his cloning work in TAV2b project; Dr Tang Xuhua, Dr. Huang jinshan, Mr. Machida for their technical support, help, and friendship. I would like to extend my thanks to Ms. Qin haina, Mr. LiuMing, and Ms. SongYan, for their sincerity and friendship. Finally but most importantly, none of my achievement is possible without the love of my family, the constant source of strength in all my life. I would like to express my heart-felt gratitude to my parents and my younger brother for their selfless love and the spiritual support all the way. Thanks especially go to my husband, Zheng Yi, for his endless love, tolerance, and encouragement.   IV    Table of Contents CHAPTER ONE: LITERATURE REVIEW 1  Part І: A Structural Perspective of the Protein–RNA Interactions Involved in Virus-induced RNA Silencing and Its Suppression 1  Summary 1  1.  Introduction . 1  2.  Key components in RNA silencing pathway . 3  2.1.  Triggers for RNA silencing 3  2.1.1.  siRNAs 3  2.1.2.  miRNAs 3  2.1.3.  piRNAs . 9  2.2.  Dicers 9  2.2.1.  Roles of Dicers in processing small RNAs 9  2.2.2.  Roles of Dicers in processing Virus-derived small interfering RNAs (viRNAs) . 10  2.2.3.  Ribonuclease III enzymes partners 10  2.2.4.  The structural understanding of Ribonuclease III family enzymes . 11  2.3.  Argonautes 15  2.3.1.  Minimal RISC . 15  2.3.2.  Argonautes partners 16  2.3.3.  P bodies . 17  2.3.4.  RISC loading complex . 18  2.3.5.  Structural understanding of Argonautes . 19  2.3.5.1.  PAZ domain 19  2.3.5.2.  Mid/PIWI domain . 20  2.3.5.3.  Structural insights into Argonaute-mediated mRNA cleavage . 20  3.  Diversity of viral suppressors of RNA silencing . 21  3.1.  An RNA silencing suppressor encoded by plant virus 25  3.1.1.  The structure of P19, an RNA silencing suppressor encoded by a plant virus . 25  3.2.  RNA silencing suppressors encoded by animal viruses 25  3.2.1.  The structure of B2, an RNA silencing suppressor encoded by an animal virus 25  3.2.2.  The structure of NS1A, an RNA silencing suppressor ebcided by an animal virus . 27  4.  Future Prospective 28  Part II: Overview of X-Ray Crystallography . 30  Summary 30  1.  Introduction . 30  2.  History . 31  3.  Crystals . 32  4.  X-ray Diffraction 33  5.  Data collection 35  6.  Structure Determination . 36  6.1.  Direct method . 36  6.2.  Molecular Replacement (MR) 37  6.3.  Isomorphous replacement method . 37  7.  Conclusions . 39  Objectives of the Projects . 40    V    Significance of the Projects 40  CHAPTER TWO: MATERIALS AND METHODS . 42  1.  Bacterial strains and media . 42  2.  Plant materials and Argro-infiltration 42  2.1.  Maintenance of plant material. . 42  2.2.  Argro-infiltration. . 42  3.  DNA manipulation . 43  3.1.  Amplification of DNA by polymerase chain reaction (PCR) 43  3.2.  Agarose gel electrophoresis and DNA purification . 43  3.3.  DNA digestion and ligation 45  3.4.  Preparation of E.coli competent cells . 45  3.5.  Transformation of bacterial cells 46  3.6.  Purification of plasmids from bacteria . 46  3.7.  Screening of transformants by restriction digestion and DNA sequencing 47  3.8.  DNA sequencing . 47  3.9.  Site-directed mutagenesis . 47  4.  Protein manipulation . 48  4.1.  Protein expression and solubility test . 48  4.2.  Expression of Seleno-Methionine substituted protein . 48  4.3.  Protein Purification . 49  4.3.1.  Protein purification by Affinity chromatography 49  4.3.1.1.  GST fusion protein purification and removal of GST tag . 49  4.3.1.2.  Polyhistidine (HIS) fusion proteins or HIS-SUMO fusion protein purification and removal of HIS or HIS-SUMO tags . 50  4.3.1.3.  Heparin affinity chromatography . 51  4.3.1.4.  Protein purification by ion exchange chromatography 53  4.3.1.5.  Gel filtration 53  5.  Crystallization . 53  6.  Data collection and structure determination 54  7.  Protein analysis . 54  7.1.  SDS-PAGE gel . 54  7.2.  Flag affinity Pull down assay . 56  7.3.  Western blotting 57  7.4.  Electrophoretic Mobility-shift assay (EMSA) . 57  7.5.  Analytical gel filtration . 58  7.6.  Isothermal Titration Calorimetry (ITC) . 59  CHAPTER THREE: CHARACTERIZATION OF KIAA1093 FUNCTIONS IN RISC THROUGH ITS C-TERMINAL RNA RECOGNITION MOTIF . 61  Summary 61  1.  Introduction . 61  2.  Results . 64  2.1.  Bioinformatics analysis of kiaa1093 RRM 64  2.2.  Native and Semet- RRM proteins purification and crystallization . 65  2.3.  Data collection and structure determination 67  2.4.  Overview structure of RRM domain 69  2.5.  Kiaa1093 RRM has no interaction with small RNA or DNA . 69  2.6.  Bioinformatics analysis of RRM binding partners 72  2.7.  RRM interacts with TRBP . 72    VI    2.7.1.  2.7.2.  2.7.3.  3.  RRM interacts with TRBP mainly via domain . 72  RRM’s C-terminal α-helix plays important role in the interaction between TRBP and RRM. . 76  The kiaa1093 RRM enhances the binding affinity between TRBP D1+2 and 21siRNA. 76  Discussion . 83  CHAPTER FOUR: STRUCTURAL BASIS FOR RNA-SILENCING SUPPRESSION BY TOMATO ASPERMY VIRUS PROTEIN 2B . 88  Summary 88  1.  Introduction . 88  2.  Results . 89  2.1.  TAV2b is a small dsRNA-binding protein 89  2.2.  TAV2b forms dimers in solution . 91  2.3.  Protein crystallization, data collection, and structural determination (This part of work is done by Chen Hongying) . 91  2.4.  Overview of the TAV2b-siRNA duplex complex structure 93  2.5.  Key residues at both RNA-protein interface and protein-protein interface of TAV2b 95  2.6.  TAV2b suppresses RNA silencing . 104  2.7.  TAV2b distinguish dsRNA from dsDNA on the basis of the major groove structure . 106  3.  Discussion . 106  CHAPTER FIVE: CONCLUSIONS 112  BIBLIOGRAPHY . 118  LIST OF PUBLICATIONS 129          VII    List of Figures Figure 1‐1: Schematic overview of siRNA pathway   4  Figure 1-2 . Schematic overview of miRNA pathway.   7  Figure 1-3. Domain arrangement of RNase III type enzymes and their structures.  . 14  Figure 1-4 . Domain arrangement of Argonautes and their structures.   22  Figure 1-5 . Molecular mechanisms of viral suppressors targeting RNA for RNA silencing suppression  . 26    Figure 3-1. Bioinformatics analysis of kiaa1093   66  Figure 3-2. Protein purification and crystallization  . 68  Figure 3-3. Structure determination of kiaa1093 RRM  . 71  Figure 3‐4. Both RRM and RRM Δ C- α helix have no interaction with the selected RNA and DNA  . 73  Figure 3‐5. Bioinformatics analysis of TRBP and its domain arrangement.   75  Figure 3‐6. Physical association of TRBP and kiaa1093 RRM  . 77  Figure 3‐7. C-terminal α helix plays important role in the interaction of kiaa1093 RRM and TRBP   . 79  Figure 3‐8. The kiaa1093 RRM might have effects on TRBP RNA binding affinity.  . 81  Figure 3‐9.The hypotheses of binding mode of TRBP, kiaa1093RRM, and Dicer   86    Figure 4‐1. TAV2b is a dsRNA binding protein.  . 90  Figure 4‐2. TAV2b forms tetramer in solution.  . 92  Figure 4‐3. Overview of TAV2b/siRNA structure  . 96  Figure 4‐4. Characterization of the RNA–protein interface and protein–protein interface of TAV2b  . 99  Figure 4‐5. ITC data of TAV2b and its mutants binding with 21nt siRNA duplex.   102    VIII    Figure 4‐6. RNA-silencing suppression in Nicotiana benthamiana (16c) by TAV2b  . 105  Figure 4‐7. TAV2b prefers to bind to dsRNA   107  Figure 4‐8. Diagram of the RNA-silencing pathway  . 111    IX    List of Table Table 2-1. Primers’ sequences used in this thesis.  . 44  Table 2-2. The general techniques of protein purification used in this thesis.  . 52  Table 2-3. SDS-PAGE gel formula.   55  Table 2-4. Small RNAs and DNAs sequences used in this thesis   60    Table 3-1. Data collection, phasing and refinement   70  Table 3‐2. TRBP different domains interact with kiaa1093 RRM  . 78    Table 4‐1. Data collection, phasing and refinement statistics.  . 94  Table 4‐2 . Key residues deferred from the TAV2b/siRNA complex structure.   100  Table 4‐3. Binding of TAV2b and its mutants with a 21-nt siRNA duplex.   103  Table 4‐4. RNA substrate recognition preference by TAV2b.  . 108    x    List of Abbreviations   AGO Argonaute bp base pair CIRV Carnation Italian ringspot virus CMV Cucumber Mosaic virus CTV Citrus Tristeza virus CV column volume Dcr1 Dicer-1 DCL Dicer like protein dsRBD double stranded RNA binding domain dsRNA double stranded RNA DUF 283 domain with unknown function EB ethidium bromide EGS Ethylene glycolbis FHV flockhouse virus GFP green fluorescent protein HRP horseradish peroxidase IPTG isopropyl-ß-D-thiogalactopyranoside LMB leptomycin B MAD multiwavelength anomalous dispersion method MIR multiple isomorphous replacement method miRNAs microRNAs MR molecular replacement method NLS nuclear location signal   RRM. The association of Dicer helicase and/or kiaa1093 RRM changes TRBP conformation, and correspondingly exposes the RNA binding domain in D2. Therefore TRBP shows higher RNA binding affinity at the presents of TRBP’s binding partners. Considering that it is D1+2 but not full length TRBP has enhanced RNA binding affinity at the present of kiaa1093 RRM, it is reasonable to hypothesize that the interaction between D3 and Dicer helicase together with interaction between D1 and kiaa1093 might completely activate the conformation change of TRBP, and sequentially strengthens the TRBP/siRNA complex. Therefore, it is possible that kiaa1093 might serve as a scaffold protein to strengthen Argonaute/Dicer/TRBP /bound siRNA supercomplex. Kiaa1093 binds with TRBP via the bridge between Cterminal RRM domain and TRBP D1 to stabilize the TRBP/siRNA interaction to facilitate the production of siRNA with Dicer. On the other hand, kiaa1093 interacts with AGOs to assist the small RNAs loading into AGOs. However, we only establish the interaction between kiaa1093 RRM and TRBP in vitro, and lack of in vivo evidence. In future, we plan to perform in vivo experiments to support our conclusions. In chapter 4, we report the complex crystal structure of TAV2b bound to a 21 siRNA duplex, which thoroughly elucidates 2b's possible strategy to suppress RNA silencing by targeting dsRNA. Viruses encode a wide range of suppressors with various sequences, motifs and structures to counter host defense by targeting different steps of RNA silencing pathway via different strategies. On the basis of the current studies, there are mainly       two strategies displayed by viral suppressors. One is targeting small RNAs, the other is to interfere with protein components in RNA silencing pathway (detailed discussion in chapter 1). Interestingly, even in the same group, the suppression mechanisms vary greatly by different suppressors. Although its homolog CMV 2b is targeting AGO1 in Arabidopsis, TAV2b is a RNA binding protein. Compared with the previous reports of suppressor/RNA complex, (either TBSV p19 which sequesters siRNA in sequence-depending mode, or FHV B2 which adopts sequence-independing mode), TAV2b adopts a novel binding mode: an all α-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. Although dimerized TAV2b also contains a pair of Trp residues projecting from each C-terminal α2 helix to recognize both ends of siRNA, the measurement of TAV2b is different from that of TBSV p19. p19 forms a head to tail homodimer arrangement and there are two sets of tryptophan residues projected form its ‘read head’ α helix to stack over the 5’-end bases of siRNA duplex, leading to effective measurement of the duplex length [104, 111]. TAV2b recognize and interact with siRNA by measuring the width of the major groove of siRNA duplex. Compared to DNAs which general adopt B-form (10.5 bp per turn), most of RNAs adopt A form, a wider right-handed spiral (general 11bp per turn). Derived from the structural information, TAV2b distinguishes dsRNAs by fitting its α-helical backbone into the major groove (Figure. 4-2). Therefore TAV2b has very low binding affinity with 7bp dsRNA and 21ssRNA, which does not form complete major grooves. And TAV2b has lower binding affinity with dsDNA (B-form) and 12nt small RNA (hardly to form a       complete turn) (Figure.4-7 and Table 4-4). Thus the measurement by TAV2b is not strictly despite of the two Trps projecting from both sides of the structure. The promiscuous structural and functional deviation of dsRNA binding by TAV2b suggests that both longer dsRNA and siRNA duplex might be the targets of TAV2b, which is similar to FHV B2 protein. We also characterize the key residues of TAV2b involved in either proteinprotein or protein-RNA interaction. The complex structure indicates that dimerized TAV2b further forms a tetramer through the conserved leucine-zipper-like motif at the N-terminal α helix (Figure 4-5B, and Table 4-2). The mutations of leucine-zipperlike motif disrupt suppression of silencing of TAV2b (Figure. 4-6). Another key residue is Try 50 which has been mentioned above. Although the pair of Trp (50) residues are not an efficient ruler for measuring the length of the bound RNA and the mutations of Trp 50 only compromise slight decrease in RNA binding affinity (Figure. 4-5E, F, and Table. 4-3), the mutations of Trp 50 greatly diminish the suppression of silencing by TAV2b (Figure. 4-6). It is possible that Trp 50 plays a role in the interaction between TAV2b and the proteins involved in the silencing pathway. TAV2b also encodes a putative NLS at the N-terminal half of the protein. Moreover, similar to CMV 2b, transient expression of TAV2b is targeted in nuclei. It was reported that CMV 2b is localized in the nuclei of tobacco suspension cells and whole plant via an arginine-rich NLS and the nuclear targeting of the CMV 2b is required for the efficient suppression of PTGS [146]. Thus it is possible that NLS encoded by TAV2b is also important for suppression of PTGS. In our report, the       mutations of Arg 28 and the invariable His 29, which are the residues in TAV2b NLS, decrease the RNA the RNA binding affinity by ten times, and are defective in silencing suppression of TAV2b in vitro. It would therefore appear that 2b proteins encoded by the cucumovirus are likely to be targeted to the nuclei of the invaded cells and PTGS may be blocked in nucleus. Previously, we have discussed there are mainly two kinds of suppressors on the basis of the current reports; one is targeted at RNA whereas the other is targeted at protein effectors in RNA silencing pathway. Interestingly, CMV2b is able to target Arabidopsis AGO1 to suppress host RNA silencing, whereas, TAV2b, another member of the cucumovirus family, is able to bind directly to siRNA duplex to repress host defense. Taken together, cucumovirus 2b proteins targets both the triggers and the silencers in RNA silencing pathway, which provides an enormous advantage for the virus to survive during the arms race against the host.     118    Bibliography 1. S. A. Wingard, Hosts and symptoms of ring spot, a virus disease of plants, J. Agric. Res. 37 (1928) 127–153. 2. D. Baulcombe, RNA silencing in plants, Nature 431 (2004) 356-363. 3. A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, C. C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (1998) 744-745. 4. E. Bernstein, A. A. Caudy, S. M. Hammond, G. J. Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409 (2001) 363–366. 5. A. Nykanen, B. Haley, P. D. Zamore, ATP requirements and small interfering RNA structure in the RNA interference pathway, Cell 107 (2001) 309–321. 6. S. M. Hammond, E. Bernstein, D. Beach, G. J. Hannon, An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cell extracts, Nature 404 (2000) 293–296. 7. J. Liu, M. A. Carmell, F. V. Rivas, C. G. Marsden, J. M. Thomson, J. J. Song, S. M. Hammond, L. Joshua-Tor, G. J. Hannon, Argonaute2 is the catalytic engine of mammalian RNAi, Science 305 (2004) 1437–1441. 8. G. Meister, M. Landthaler, A, Patkaniowska, Y. Dorsett, G. Teng, T. Tuschl, Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs, Mol. Cell 15 (2004) 185-197. 9. H. Siomi, M. C. Siomi, On the road to reading the RNA-interference code, Nature 457 (2009) 396-404. 10. O. Voinnet, Induction and suppression of RNA silencing: insights from viral infections, Nature (2005) 206-220. 11. S. W. Ding, O. Voinnet, Antiviral Immunity Directed by Small RNAs, Cell 130 (2007) 413-426. 12. O. Voinnet, Use, tolerance and avoidance of amplified RNA silencing by plants, Trends Plant Sci. 13 (2008) 317-328. 13. C. Lacomme, K. Hrubikova, I. Hein, Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats, Plant J. 34 (2003) 543–553. 14. A. Hamilton, O. Voinnet, L. Chappell, D. Baulcombe, Two classes of short interfering RNA in RNA silencing, EMBO J. 21 (2002) 4671–4679. 15. T. A. Farazi, S. A. Juranek, T. Tuschl, The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members, Development 135 (2008) 1201-1214. 16. A. A. Aravin, M. Lagos-Quintana, A. Yalcin, M. Zavolan, D. Marks, B, Snyder, T. Gaasterland, J. Meyer, T. Tuschl, The small RNA profile during Drosophila elanogaster development, Dev. Cell (2003) 337-350.   119    17. Y. Lee, K. Jeon, J. T. Lee, S. Kim, V. N. Kim, MicroRNA maturation: stepwise processing and subcellular localization, EMBO J. 21 (2002) 4663-4670. 18. V. N. Kim, MicroRNA precursors in motion: exportin-5 mediates their nuclear export, Trends Cell Biol. 14 (2004) 156-159. 19. D. S. Schwarz, G. Hutvagner, T. Du, Z. Xu, N. Aronin, P. D. Zamore, Asymmetry in the assembly of the RNAi enzyme complex, Cell 115 (2003) 199-208. 20. B. Yu, Z. Yang, J. Li, S. Minakhina, M. Yang, R. W. Padgett, R. Steward, X. Chen, Methylation as a crucial step in plant microRNA biogenesis, Science 307 (2005) 932935. 21. X. Chen, MicroRNA metabolism in plants, Curr. Top. Microbiol. Immunol. 320 (2008) 117–136. 22. K. Okamura, A. Ishizuka, H. Siomi, M. C. Siomi, Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways, Genes Dev. 18 (2004) 1655-1666. 23. L. Peters, G. Meister, Argonaute proteins: mediators of RNA silencing, Mol. Cell 26 (2007) 611-623. 24. S. Pfeffer, M. Zavolan, F. A. Grässer, M. Chien, J. J. Russo, J. Ju, B. John, A. J. Enright, D. Marks, C. Sander, T. Tuschl, Identification of virus-encoded microRNAs, Science 304 (2004) 734-736. 25. E. Murphy, J. Vanícek, H. Robins, T. Shenk, A. J. Levine, Suppression of immediateearly viral gene expression by herpesvirus-coded microRNAs: implications for latency, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 5453-5458. 26. B. R. Cullen, Viral and cellular messenger RNA targets of viral microRNAs, Nature 457 (2009) 421-425. 27. V. V. Vagin, A. Sigova, C. Li, H. Seitz, V. Gvozdev, P. D. Zamore, A distinct small RNA pathway silences selfish genetic elements in the germline, Science 313 (2006) 320-324. 28. J. Brennecke, A. A. Aravin, A. Stark, M. Dus, M. Kellis, R. Sachidanandam, G. J. Hannon, Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila, Cell 128 (2007) 1089-1103. 29. K. Saito, Y. Sakaguchi, T. Suzuki, T. Suzuki, H. Siomi, M. C. Siomi, Pimet, the Drosophila homolog of HEN1, mediates 2’-O-methylation of Piwi- interacting RNAs at their 3’ ends, Genes Dev. 21 (2007) 1603-1608. 30. T. Ohara, Y. Sakaguchi, T. Suzuki, H. Ueda, K. Miyauchi, T. Suzuki, The 3’termini of mouse Piwi-interacting RNAs are 2’-O-methylated, Nat. Struct. Mol. Biol. 14 (2007) 349-350. 31. Y. Kirino, Z. Mourelatos, The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs, RNA 13 (2007) 1397-1401. 32. Y. Kirino, Z. Mourelatos, Mouse Piwi-interacting RNAs are 2’-O-methylated at their 3’termini, Nat. Struct. Mol. Biol. 14 (2007) 347-348.   120    33. M. D. Horwich, C. Li, C. Matranga, V. Vagin, G. Farley, P. Wang, P. D. Zamore, The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and singlestranded siRNAs in RISC, Curr. Biol. 17 (2007) 1265-1272. 34. K. Saito, A. Ishizuka, H. Siomi, M. C. Siomi, Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells, PLoS Biol. (2005) e235. 35. X. Liu, J. K. Park, F. Jiang, Y. Liu, D. McKearin, Q. Liu. Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes, RNA 13 (2007) 2324-2329. 36. S. M. Hammond, Dicing and slicing: the core machinery of the RNA interference pathway, FEBS Lett. 579 (2005) 5822–5829. 37. P. Dunoyer, C. Himber, O. Voinnet, DICER-LIKE is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-tocell silencing signal, Nat. Genet. 37 (2005) 1356–1360. 38. V. Gasciolli, A. C. Mallory, D. P. Bartel, H. Vaucheret, Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs, Curr. Biol. 15 (2005) 1494–1500. 39. Y. Qi, A. M. Denli, G. J. Hannon, Biochemical specialization within Arabidopsis RNA silencing pathways, Mol. Cell 19 (2005) 421–428. 40. Z. Xie, L. K. Johansen, A. M. Gustafson, K. D. Kasschau, A. D. Lellis, D. Zilberman, S. E. Jacobsen, J. C. Carrington, Genetic and functional diversification of small RNA pathways in plants, PLoS Biol. (2004) E104. 41. X. H. Wang, R. Aliyari, W. X. Li, H. W. Li, K. Kim, R. Carthew, P. Atkinson, S. W. Ding, RNA interference directs innate immunity against viruses in adult Drosophila, Science 312 (2006) 452-454. 42. A. Deleris, J. Gallego-Bartolome, J. Bao, K. D. Kasschau, J. C. Carrington, O. Voinnet, Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense, Science 313 (2006) 68–71. 43. N. Bouche, D. Lauressergues, V. Gasciolli, H. Vaucheret, An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs, EMBO J. 25 (2006) 3347–3356. 44. J. Han, Y. Lee, K. H. Yeom, J. W. Nam, I. Heo, J. K. Rhee, S. Y. Sohn, Y. Cho, B. T. Zhang, V. N. Kim, Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex, Cell 125 (2006) 887-901. 45. Q. Liu , T. A. Rand, S. Kalidas, F. Du, H. E. Kim, D. P. Smith, X. Wang, R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway, Science 301 (2003) 1921–1925. 46. Y. Tomari, C. Matranga, B. Haley, N. Martinez, P. D. Zamore, A protein sensor for siRNA asymmetry, Science 306 (2004) 1377-1380. 47. S. Y. Sohn, W. J. Bae, J. J. Kim, K. H. Yeom, V. N. Kim, Y. Cho, Crystal structure of human DGCR8 core, Nat. Struct. Mol. Biol. 14 (2007) 847-853.   121    48. Y. Kurihara, Y. Takashi, Y. Watanabe, The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis, RNA 12 (2006) 206-212. 49. Z, Dong, M. H. Han, N. Fedoroff, The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 9970-9975. 50. J. Gan, J. E. Tropea, B. P. Austin, D. L. Court, D. S. Waugh, X. Ji, Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III, Cell 124 (2006) 355-366. 51. H. Zhang, F. A. Kolb, L. Jaskiewicz, E. Westhof, W. Filipowicz, Single processing center models for human Dicer and bacterial RNase III, Cell 118 (2004) 57-68. 52. I. J. Macrae, K. Zhou, F. Li, A. Repic, A. N. Brooks, W. Z. Cande, P. D. Adams, J. A. Doudna, Structural basis for double-stranded RNA processing by Dicer, Science 311 (2006) 195-198. 53. J. J. Song , J. Liu, N. H. Tolia, J. Schneiderman, S. K. Smith, R. A. Martienssen, G. J. Hannon, L. Joshua-Tor, The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes, Nat. Struct. Biol. 10 (2003) 1026–1032. 54. A. Lingel, B. Simon, E. Izaurralde, M. Sattler, Structure and nucleic-acid binding of the Drosophila Argonaute PAZ domain, Nature 426 (2003) 465–469. 55. K. S. Yan, S. Yan, A. Farooq, A. Han, L. Zeng, M. M. Zhou, Structure and conserved RNA binding of the PAZ domain, Nature 426 (2003) 468-474. 56. J. B. Ma, K. Ye, D. J. Patel, Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain, Nature 429 (2004) 318-322. 57. Z. Du, J. K. Lee, R. Tjhen, R. M. Stroud, T. L. James, Structural and biochemical insights into the dicing mechanism of mouse Dicer: a conserved lysine is critical for dsRNA cleavage, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 2391-2396. 58. M. Dlakić, DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold, Bioinformatics 22 (2006) 2711-2714. 59. H. Zhang, F. A. Kolb, V. Brondani, E. Billy, W. Filipowicz, Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP, EMBO J. 21 (2002) 5875-5885. 60. P. Provost, D. Dishart, J. Doucet, D. Frendewey, B. Samuelsson, O. Rådmark, Ribonuclease activity and RNA binding of recombinant human Dicer, EMBO J. 21 (2002) 5864-5874. 61. J. W. Pham, J. L. Pellino, Y. S. Lee , R. W. Carthew, E. J. Sontheimer, A Dicer-2dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila, Cell 117 (2004) 83-94. 62. S. U. Colmenares, S. M. Buker, M. Buhler, M. Dlakic, D. Moazed, Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi, Mol. Cell 27 (2007) 449-461.   122    63. Y. Tagami, H. Motose, Y. Watanabe, A dominant mutation in DCL1 suppresses the hyl1 mutant phenotype by promoting the processing of miRNA, RNA 15 (2009) 450458. 64. E. Ma, I. J. MacRae, J. F. Kirsch, J. A. Doudna, Autoinhibition of human dicer by its internal helicase domain, J. Mol. Biol. 380 (2008) 237-243. 65. J. S. Parker, D. Barford, Argonaute: A scaffold for the function of short regulatory RNAs, Trends Biochem. Sci. 31 (2006) 622-630. 66. K. Miyoshi, H. Tsukumo, T. Nagami, H, Siomi, M. C. Siomi, Slicer function of Drosophila Argonautes and its involvement in RISC formation, Genes Dev. 19 (2005) 2837–2848. 67. F. V. Rivas, N. H. Tolia, J. J. Song, J. P. Aragon, J. Liu, G. J. Hannon, L. Joshua-Tor, Purified Argonaute2 and an siRNA form recombinant human RISC, Nat. Struct. Mol. Biol. 12 (2005) 340–349. 68. J. J. Song, S. K. Smith, G. J. Hannon, L. Joshua-Tor, Crystal structure of Argonaute and its implications for RISC slicer activity, Science 305 (2004) 1434-1437. 69. G. Hutvagner, M. J. Simard, Argonaute proteins: key players in RNA silencing, Nat. Rev. Mol. Cell Biol. (2008) 22-32. 70. S. Mi, T. Cai, Y. Hu, Y. Chen, E. Hodges, F. Ni, L. Wu, S. Li, H. Zhou, C. Long, S. Chen, G. J. Hannon, Y. Qi, Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5’ terminal nucleotide, Cell 133 (2008) 116-127. 71. X. Zhang, Y. R. Yuan, Y. Pei, S. S. Lin, T. Tuschl, D. J. Patel, N. H. Chua, Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute cleavage activity to counter plant defence, Genes Dev. 20 (2006) 3255-3268. 72. T. P. Chendrimada, R. I. Gregory, E. Kumaraswamy, J. Norman, N. Cooch, K. Nishikura, R. Shiekhattar, TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing, Nature 436 (2005) 740-744. 73. L. Weinmann, J. Höck, T. Ivacevic, T. Ohrt, J. Mütze, P. Schwille, E. Kremmer, V. Benes, H. Urlaub, G. Meister, Importin is a gene silencing factor that targets Argonaute proteins to distinct mRNAs, Cell 136 (2009) 496-507. 74. J. Höck, L. Weinmann, C. Ender, S. Rüdel, E. Kremmer, M. Raabe, H. Urlaub, G. Meister, Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells, EMBO Rep. (2007) 1052-1060. 75. S. M. Hammond, S. Boettcher, A. A. Caudy, R. Kobayashi, G. J. Hannon, Argonaute2, a link between genetic and biochemical analyses of RNAi, Science 293 (2001) 1146-1150. 76. N. Tahbaz, F. A. Kolb, H. Zhang, K. Jaronczyk, W. Filipowicz, T. C. Hobman, Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer, EMBO Rep. (2004) 189-194. 77. G. Meister, M. Landthaler, L. Peters, P. Y. Chen, H. Urlaub, R. Lűhrmann, T. Tuschl, Identification of Novel Argonaute-Associated Proteins, Curr. Biol. 15 (2005) 2149– 2155.   123    78. G. B. Robb, T. M. Rana, RNA helicase A interacts with RISC in human cells and functions in RISC loading, Mol. Cell 26 (2007) 523-537. 79. T. Eystathioy, E. K. Chan, S. A. Tenenbaum, J. D. Keene, K. Griffith, M. J. Fritzler, A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles, Mol. Biol. Cell 13 (2002) 1338–1351. 80. A. Jakymiw, S. Lian, T. Eystathioy, S. Li, M. Satoh, J. C. Hamel, M. J. Fritzler, E. K. Chan, Disruption of GW bodies impairs mammalian RNA interference, Nat. Cell Biol. (2005) 1267-1274. 81. J. Liu, F. W. Rivas, J. Wohlschlegel, J. R. III. Yates, R. Parker, G. J. Hannon, A role for the P-body component GW182 in microRNA function, Nat. Cell Biol. (2005) 1261-1266. 82. G. L. Sen, H. M. Blau, Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies, Nat. Cell Biol. (2005) 633–636. 83. S. Till, E. Lejeune, R. Thermann, M. Bortfeld, M. Hothorn, D. Enderle, C. Heinrich, M. W. Hentze, A. G. Ladurner, A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain, Nat. Struct. Mol. Biol. 14 (2007) 897-903. 84. A. Eulalio, E. Huntzinger, E, Izaurralde, GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay, Nat. Struct. Mol. Biol. 15 (2008) 346-353. 85. A. Eulalio, I. Behm-Ansmant, E. Izaurralde, P bodies: at the crossroads of posttranscriptional pathways, Nat. Rev. Mol. Cell Biol. (2007) 9-22. 86. C. Y. Chu, T. M. Rana, Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54, PLoS Biol. (2006) e210. 87. J. Rehwinkel, I. Behm-Ansmant, D. Gatfield, E. Izaurralde, A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing, RNA 11 (2005) 1640–1647. 88. A. Eulalio, I. Behm-Ansmant, D. Schweizer, E. Izaurralde, P-body formation is a consequence, not the cause, of RNA-mediated gene silencing, Mol. Cell Biol. 27 (2007) 3970-3981. 89. M. She, C. J. Decker, D. I. Svergun, A. Round, N. Chen, D. Muhlrad, R. Parker, H. Song, Structural basis of Dcp2 recognition and activation by Dcp1, Mol. Cell 29 (2008) 337–349. 90. M. Landthaler, D. Gaidatzis, A. Rothballer, P. Y. Chen, S. J. Soll, L. Dinic, T. Ojo, M. Hafner, M. Zavolan, T. Tuschl, Molecular characterization of human Argonautecontaining ribonucleoprotein complexes and their bound target mRNAs, RNA 14 (2008) 2580-2596. 91. C. Matranga, Y. Tomari, C, Shin, D. P. Bartel, P. D. Zamore, Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes, Cell 123 (2005) 607-620. 92. F. Jiang, X. Ye, X. Liu, L. Fincher, D. McKearin, Q. Liu, Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila, Genes Dev.19 (2005) 1674-1679.   124    93. Y. Lee, I. Hur, S. Y. Park, Y. K. Kim, M. R. Suh, V. N. Kim, The role of PACT in the RNA silencing pathway, EMBO J. 25 (2006) 522-532. 94. R. I. Gregory, T. P. Chendrimada, N. Cooch, R. Shiekhattar, Human RISC couples microRNA biogenesis and posttranscriptional gene silencing, Cell 123 (2005), 631– 640. 95. J. S. Parker, S. M. Roe, D. Barford, Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex, Nature 434 (2005) 663-666. 96. J. B. Ma, Y. R. Yuan, G. Meister, Y. Pei, T. Tuschl, D. J. Patel, Structural basis for 5'end-specific recognition of guide RNA by the A. fulgidus Piwi protein, Nature 434 (2005) 666-670. 97. Y. R. Yuan, Y. Pei, J. B. Ma, V. Kuryavyi, M. Zhadina, G. Meister, H. Y. Chen, Z. Dauter, T. Tuschl, D. J. Patel, Crystal structure of A. aeolicus argonaute, a sitespecific DNA-guided endoribonuclease, provides insights into RISC mediated mRNA cleavage, Mol. Cell 19 (2005) 405–419. 98. Y. Wang, S. Juranek, H. Li, G. Sheng, T. Tuschl, D. J. Patel, Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex, Nature 456 (2008) 921-926. 99. M. Nowotny, S. A. Gaidamakov, R. J. Crouch, W. Yang, Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis, Cell 121 (2005) 1005-1016. 100. Y. R. Yuan, Y. Pei, H. Y. Chen, T. Tuschl, D. J. Patel, A potential protein-RNA recognition event along the RISC-loading pathway from the structure of A. aeolicus Argonaute with externally bound siRNA, Structure 14 (2006) 1557-1565. 101. U. J. Rashid, D. Paterok, A. Koglin, H. Gohlke, J. Piehler, J. C. Chen, Structure of Aquifex aeolicus argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function, J. Biol. Chem. 282 (2007) 13824-13832. 102. Y. Wang, G. Sheng, S. Juranek, T. Tuschl, D. J. Patel, Structure of the guide-strandcontaining Argonaute silencing complex, Nature 456 (2008) 209-213. 103. A. C. Mallory, B. J. Reinhart, D. Bartel, V. B. Vance, L. H. Bowman, A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 15228-15233. 104. J. M. Vargason, G. Szittya, J. Burgyăn, T. M. Hall, Size Selective Recognition of siRNA by an RNA Silencing Suppressor, Cell 115 (2003) 799-811. 105. N. Baumberger, C. H. Tsai, M. Lie, E. Havecker, D. C. Baulcombe, The Polerovirus silencing suppressor P0 targets Argonaute proteins for degradation, Curr. Biol. 17 (2007) 1609-1614. 106. D. Bortolamiol, M. Pazhouhandeh, K. Marrocco, P. Genschik, V. Ziegler-Graff, The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing, Curr. Biol. 17 (2007) 1615-1621. 107. M. Pazhouhandeh, M. Dieterle, K. Marrocco, E. Lechner, B. Berry, V. Brault, O. Hemmer, T. Kretsch, K. E. Richards, P. Genschik, V. Ziegler-Graff, F-box-like   125    domain in the polerovirus protein P0 is required for silencing suppressor function, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 1994-1999. 108. A. Levy, M. Dafny-Yelin, T. Tzfira, Attacking the defenders: plant viruses fight back. Trends Microbiol. 16 (2008) 194-197. 109. R. Lu, A. Folimonov, M. Shintaku, W. X. Li, B. W. Falk, W. O. Dawson, S. W. Ding, Three distinct suppressors of RNA silencing encoded by a 20-kb viral genome, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 15742-15747. 110. C. López, J. Navas-Castillo, S. Gowda, P. Moreno, R. Flores, The 23 –kD protein coded by the 3’- terminal gene of Citrus Tristeza Virus is an RNA binding protein, Virology 269 (2000) 462-470. 111. K. Ye, L. Malinina, D. J. Patel, Recognition of small interfering RNA by a viral suppressor of RNA silencing, Nature 426 (2003) 874-878. 112. J. A. Chao, J. H. Lee, B. R. Chapados, E. W. Debler, A. Schneemann, J. R. Williamson, Dual modes of RNA-silencing suppression by Flock House virus protein B2, Nat. Struct. Mol. Biol. 12 (2005) 952-957. 113. Lingel, B. Simon, E. Izaurralde, M. Sattler, The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of doublestranded RNA recognition, EMBO Rep.12 (2005)1149-1155. 114. D. Silhavy, A. Molnár, A, Lucioli, G. Szittya, C. Hornyik, M. Tavazza, J. Burgyán, A viral protein suppresses RNA silencing and binds silencing-generated, 21-to 25nucleotide double-stranded RNA, EMBO J. 21 (2002) 3070–3080. 115. K. N. Johnson, K. L. Johnson, R. Dasgupta, T. Gratsch, L. A. Ball, Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases, J. Gen. Virol. 82 (2001) 1855–1866. 116. H. Li, W. X. Li, S. W. Ding, Induction and suppression of RNA silencing by an animal virus, Science 296 (2002) 1319–1321. 117. Y. Qiu, R. M. Krug, The influenza virus NS1 protein is a poly(A)- binding protein that inhibits nuclear export of mRNAs containing poly(A), J. Virol. 68 (1994) 24252432. 118. Y. Lu, M. Wambach, M. G. Katze, R. M. Krug, Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor, Virology 214 (1995) 222-228. 119. M. Bergmann, A. Garcia-Sastre, E. Carnero, H. Pehamberger, K. Wolff, P. Palese, T. Muster, Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication, J. Virol. 74 (2000) 6203-6206. 120. J. Y. Min, S. Li, G. C. Sen, R. M. Krug, A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis, Virology 363 (2007) 236-243. 121. C. Y. Chien, R. Tejero, Y. Huang, D. E. Zimmerman, C. B. Ríos, R. M. Krug, G. T. Montelione, A novel RNA-binding motif in influenza A virus non-structural protein 1. Nat. Struct. Biol. (1997) 891-895.   126    122. J. Liu, P. A. Lynch, C. Y. Chien, G. T. Montelione, R. M. Krug, H. M. Berman, Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein, Nat. Struct. Biol. (1997) 896-899. 123. C. Yin, J. A. Khan, G. V. Swapna, A. Ertekin, R. M. Krug, L, Tong, G. T. Montelione, Conserved surface features form the double-stranded RNA binding site of non-structural protein (NS1) from influenza A and B viruses, J. Biol. Chem. 282 (2007) 20584-20592. 124. Z. A. Bornholdt, B. V. Prasad, X-ray structure of influenza virus NS1 effector domain, Nat. Struct. Mol. Biol. 13 (2006) 559-560. 125. C. M. Newby, L. Sabin, A. Pekosz, The RNA binding domain of influenza A virus NS1 protein affects secretion of tumor necrosis factor alpha, interleukin-6, and interferon in primary murine tracheal epithelial cells, J. Virol. 81 (2007) 9469-9480. 126. A. Cheng, S. M. Wong, Y. A. Yuan, Structural basis for dsRNA recognition by NS1 protein of influenza A virus, Cell Res. 19 (2009) 187-195. 127. J. MacRae, K. Zhou, J. A. Doudna, Structural determinants of RNA recognition and cleavage by Dicer, Nat. Struct. Mol. Biol. 14 (2007) 934-940. 128. N. E. Chayen, Turning protein crystallisation from an art into a science, Curr. Opin. Struct. Biol. 14 (2004) 577-83. 129. H. Muirhead, M. Perutz, Structure of hemoglobin. A three-dimensional fourier synthesis of reduced human hemoglobin at 5.5 Å resolution, Nature 199 (1963) 633638. 130. J. Kendrew, G. Bodo, H. Dintzis, P. Parrish, H. Wyckoff, D. Phillips, A threedimensional model of the myoglobin molecule obtained by x-ray analysis, Nature (1958). 181 662-666. 131. J. Deisenhofer, O. Epp, K. Miki, R. Huber, H. Michel, Structure of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viridis at 3Å resolution, Nature 318 (1985), 618–624. 132. D. A. Doyle, J. Morais Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, R. MacKinnon, The structure of the potassium channel: molecular basis of K+ conduction and selectivity, Science 280 (1998) 69-77. 133. N. Ban, P. Nissen, J. Hansen, P. Moore, T. Steitz, The complete atomic structure of the large ribosomal subunit at 2.4 A resolution, Science 289 (2000) 905–920. 134. F. Schluenzen, A. Tocilj, R. Zarivach, J. Harms, M. Gluehmann, D. Janell, A. Bashan, H. Bartels, I. Agmon, F. Franceschi, A. Yonath, Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution, Cell 102 (2000) 615–623. 135. B. T. Wimberly, D. E. Brodersen, W. M. Clemons, R. J. Morgan-Warren, A.P. Carter, C. Vonrhein, T. Hartsch, V. Ramakrishnan, Structure of the 30S ribosomal subunit, Nature 407(2000) 327-339. 136. M. M. Yusupov, G. Z. Yusupova, A. Baucom, K. Lieberman, T. N. Earnest, J. H. Cate, H. F. Noller, Crystal structure of the ribosome at 5.5 Å resolution, Science (2001) 883-896.   127    137. J. P. Abrahams, A. G. Leslie, R. Lutter, J. E. Walker, Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria, Nature 370 (1994) 621-628. 138. J. M. Chandonia, S. E. Brenner, The impact of structural genomics: expectations and outcomes, Science 311 (2006) 347–351. 139. W. Jiang, M. L. Baker, J. Jakana, P. R. Weigele, J. King, W. Chiu, Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy, Nature 451 (2008) 1130-1134. 140. A. D. Haase, L. Jaskiewicz, H. Zhang, S. Laine, R. Sack, A. Gatignol, W. Filipowicz, TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing, EMBO Rep. (2005) 961–967. 141. A. Gatignol, A. Buckler-White, B. Berkhout, K. T. Jeang, Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR, Science 251 (1991) 1597-1600. 142. L. Daviet, M. Erard, D. Dorin, M. Duarte, C. Vaquero, A. Gatignol, Related Articles Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif, Eur. J. Biochem. 267 (2000) 24192431 143. S. M. Daniels, C. E. Melendez-Peña, R. J. Scarborough, A. Daher, H. S. Christensen, M. El Far, D. F. Purcell, S. Lainé, A. Gatignol, Characterization of the TRBP domain required for dicer interaction and function in RNA interference, BMC. Mol. Biol. (2009) 10-38. 144. G. Laraki, G. Clerzius, A. Daher, C. Melendez-Peña, S. Daniels, A. Gatignol, Interactions between the double-stranded RNA-binding proteins TRBP and PACT define the Medipal domain that mediates protein-protein interactions, RNA Biol.5 (2008) 92-103. 145. A. Cléry, M. Blatter, F. H. Allain, RNA recognition motifs: boring? Not quite, Curr. Opin. Struct. Biol. 18 (2008) 290-298. 146. R. Spadaccini, U. Reidt, O. Dybkov, C. Will, R. Frank, G. Stier, L. Corsini, M. C. Wahl, R. Lührmann, M. Sattler, Biochemical and NMR analyses of an SF3b155-p14U2AF-RNA interaction network involved in branch point definition during premRNA splicing, RNA 12 (2006) 410-425. 147. F. C. Oberstrass, S. D. Auweter, M. Erat, Y. Hargous, A. Henning, P. Wenter, L. Reymond, B. Amir-Ahmady, S. Pitsch, D. L. Black, F. H. Allain, Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation, Science 309 (2005) 2054-2057. 148. K. Goto, T. Kobori, Y. Kosaka, T. Natsuaki, C. Masuta, Characterization of silencing suppressor 2b of cucumber mosaic virus based on examination of its small RNAbinding abilities, Plant Cell Physiol. 48 (2007) 1050-1060. 149. P. Lucy, H. S. Guo, W. X. Li, S. W. Ding, Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus, EMBO J. 19 (2000) 16721680.   128    150. B. J. Shi, R. H. Symons, P. Palukaitis, The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection, Nucleic Acids Res. 36 (2008) 1057–1071. 151. K. Förstemann, Y. Tomari, T. Du, V. V. Vagin, A. M. Denli, D. P. Bratu, C. Klattenhoff, W. E. Theurkauf , P. D. Zamore, Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNAbinding domain protein, PLoS Biol. 3(2005) e236.   129    List of Publications • Chen HY, Yang J, Lin C, Yuan YA, Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b, EMBO reports (2008) 754-60 (As co-first author) • Yang J, Yuan YA. A structural perspective of the protein-RNA interactions involved in virus-induced RNA silencing and its suppression, Biochim. Biophys Acta. 2009 Jun ( Review, In press)     [...]... virus (FHV) B2   1    Chapter One: Literature Review Part І: A Structural Perspective of the Protein RNA Interactions Involved in Virus-induced RNA Silencing and Its Suppression Summary RNA silencing regulated by small RNAs, including siRNAs, miRNAs, and piRNAs, results in sequence specific inhibition of gene expression by translational repression and/ or mRNA degradation, which acts as an ancient cell... response (TAR) RNA- binding protein TRSV Tobacco Ringspot virus UV ultraviolet viRNA   Non -structural protein 1 virus-derived small interfering RNA xii    Summary RNA silencing, which is triggered by small RNAs, is a powerful gene expression regulation mechanism and results in sequence specific inhibition of gene expression by translational repression and/ or mRNA degradation Small interfering RNAs (siRNAs)... regulation of gene silencing and the cross-talk between hosts and pathogens This chapter of literature review will present current progress on the understanding of RNA silencing and especially highlight the structural principles determining the protein– RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors   3    2 Key components in RNA silencing. .. past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors This chapter will review the current understanding of the structural components in RNA silencing pathway and the structural mechanisms of RNA silencing suppression 1 Introduction RNA silencing, an RNA- based gene regulatory mechanism, is regarded... miRNAs and siRNAs, piRNAs are not processed by RNase III enzymes [27] In Drosophila, piRNA generation follows a so called “ping-pong” model with two kinds of piRNAs [28]: one is genetically encoded primary piRNAs and the other is adaptive secondary piRNAs Primary piRNAs are generated from piRNA clusters that contain the highest density of transposon-related sequences Primary piRNAs interact with and. .. pri-miRNA into pre-miRNA hairpin Drosha contains an N terminal proline-rich region, two RNase III domains in tandem and a dsRBD Drosha recognizes and processes pri-miRNA with the assistance of DGCR8 in the “ssRNAdsRNA Junction Anchoring” Model DGCR8 recognizes the stem-ssRNA junction portion of pri-miRNA and recruits Drosha to cleave the pri-miRNA around 11 bp away from the stem-ssRNA junction [44]... aberrant RNA into dsRNA, which is distinct to siRNA pathways in human and Drosophila Plant viruses encode numerous viral suppressors targeting at different steps of siRNA pathway to suppress RNA silencing For example, HcPro targets the long dsRNA; P19 targets the siRNA duplex, whereas CMV2b and P0 target AGO1 C siRNA pathway in Drosophila Dcr-2 and AGO2 are the key catalytic functional components involved... counter TRSV infection [1, 2] Despite of the early discovery, research on RNA silencing has been boomed up recently right after the discovery of double stranded RNA (dsRNA) as a trigger to activate RNA silencing [3] RNA silencing is an evolutionarily conserved process comprising a set of following core reactions Firstly, Dicer-like RNase III enzymes recognize and process long complementary dsRNA into 21-24... for RNA silencing Small dsRNAs harboring three distinct features (21-30 nucleotides (nt) in length; 5’-phosphate; and 3’-2 nt overhangs.) serve as the triggers to activate RNA silencing pathway These small dsRNAs are mainly grouped into three classes: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-associated interfering RNAs (piRNAs) 2.1.1 siRNAs siRNAs are processed from long dsRNA... produce 24 bp siRNAs and 21bp siRNAs, respectively [14, 37, 38, 39, 40] 2.2.2 Roles of Dicers in processing Virus-derived small interfering RNAs (viRNAs) Both the long dsRNA replication intermediates and the imperfect RNA hairpins derived from viral RNAs are processed into dsRNAs by RNase III enzymes to activate RNA silencing In Drosophila, Dcr-2/R2D2 heterodimer is responsible for loading viRNA into AGO2 . the current understanding of the structural components in RNA silencing pathway and the structural mechanisms of RNA silencing suppression. 1. Introduction RNA silencing, an RNA- based gene regulatory. Virus-induced RNA Silencing and Its Suppression Summary RNA silencing regulated by small RNAs, including siRNAs, miRNAs, and piRNAs, results in sequence specific inhibition of gene expression by. repression and/ or mRNA degradation. Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by RNase III enzymes and subsequently loaded into Argonaute (AGO) proteins, a key component