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  • Review Article
  • Published:

Illuminating the silence: understanding the structure and function of small RNAs

Key Points

  • RNA interference (RNAi) is an ancient and evolutionarily conserved gene-silencing mechanism that is triggered by double-stranded (ds)RNA. dsRNA, which is produced endogenously or introduced exogenously, is processed into small (21–23-nucleotide (nt)) duplexes, and the antisense strands are loaded onto RNA-induced silencing complexes (RISCs).

  • Dictated by complementarity to its target mRNA, the antisense strand guides RISC to hybridize with the target and induces gene silencing by initiating either target cleavage or assembly into RNA–protein complexes that are destined for translation suppression and localization to cytoplasmic RNA-processing foci. These two gene-silencing pathways are discussed in terms of kinetic models and the structural basis of the underlying mechanisms.

  • The major determinant of small RNAs that functions as specific triggers of gene silencing is the A-form helical geometry of RNA. The A-form helix and high-resolution structural studies provide a context for discussing the importance of phosphate groups at the 5′ end of the guide strand.

  • Small RNAs can bind to mRNAs with partial complementarity and induce silencing of unintended mRNA transcripts, known as off-target effects. Drawing on the structure and function of small RNAs and kinetic models for RNAi, this review presents plausible mechanisms for off-target effects and discusses how chemical modifications can reduce unintended gene silencing.

  • Gene silencing has been modulated and the in vivo half-life of triggers has been enhanced by effective chemical modification of RNA triggers. The review develops and discusses guidelines for the chemical modification of RNA triggers.

  • Clarifying the structure and function of the triggers of the RNAi machinery and developing RNA-delivery technologies will increase the applicability of RNAi to biology and medicine. This, in turn, will enhance our understanding of the various cellular pathways that are controlled by small RNAs.

Abstract

RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.

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Figure 1: Steps in human siRISC function.
Figure 2: A model for human miRISC function.
Figure 3: Chemical and structural features of RNA and DNA helices.
Figure 4: Structural model of a 19-nucleotide guide strand and its target RNA bound to the PIWI-domain-containing protein of Archaeoglobus fulgidus.
Figure 5: Kinetic checkpoints for RISC assembly and function.
Figure 6: Mechanism of RISC–target interactions.

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References

  1. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998). First report showing that dsRNA can silence specific genes in animals.

    CAS  PubMed  Google Scholar 

  2. Williams, B. R. Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem. Soc. Trans. 25, 509–513 (1997).

    CAS  PubMed  Google Scholar 

  3. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    CAS  PubMed  Google Scholar 

  4. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001).

    CAS  PubMed  Google Scholar 

  5. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191–3197 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    CAS  PubMed  Google Scholar 

  7. Caplen, N. J., Parrish, S., Imani, F., Fire, A. & Morgan, R. A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl Acad. Sci. USA 98, 9742–9747 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001). This paper, together with reference 7, showed that siRNA can trigger RNAi in mammalian cells.

    CAS  PubMed  Google Scholar 

  9. Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854 (1993). Discovery of the first miRNA.

    CAS  PubMed  Google Scholar 

  10. Doench, J. G., Petersen, C. P. & Sharp, P. A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Zeng, Y., Yi, R. & Cullen, B. R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl Acad. Sci. USA 100, 9779–9784 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Lippman, Z. & Martienssen, R. The role of RNA interference in heterochromatic silencing. Nature 431, 364–370 (2004).

    CAS  PubMed  Google Scholar 

  14. Waterhouse, P. M., Graham, M. W. & Wang, M. B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl Acad. Sci. USA 95, 13959–13964 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Hannon, G. J. & Rossi, J. J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378 (2004).

    CAS  PubMed  Google Scholar 

  16. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    CAS  PubMed  Google Scholar 

  17. Carmell, M. A. & Hannon, G. J. RNase III enzymes and the initiation of gene silencing. Nature Struct. Mol. Biol. 11, 214–218 (2004).

    CAS  Google Scholar 

  18. Gregory, R. I., Chendrimada, T. P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005).

    CAS  PubMed  Google Scholar 

  19. Kim, D. H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnol. 23, 222–226 (2005).

    CAS  Google Scholar 

  20. Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nature Biotechnol. 23, 227–231 (2005).

    CAS  Google Scholar 

  21. Wienholds, E. & Plasterk, R. H. MicroRNA function in animal development. FEBS Lett. 579, 5911–5922 (2005).

    CAS  PubMed  Google Scholar 

  22. Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).

    CAS  PubMed  Google Scholar 

  23. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  PubMed  Google Scholar 

  24. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    CAS  PubMed  Google Scholar 

  25. Carrington, J. C. Small RNAs and Arabidopsis. A fast forward look. Plant Physiol. 138, 565–566 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 (2001).

    CAS  PubMed  Google Scholar 

  27. Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862 (2001).

    CAS  PubMed  Google Scholar 

  28. Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kim, V. N. MicroRNA biogenesis: coordinated cropping and dicing. Nature Rev. Mol. Cell Biol. 6, 376–385 (2005).

    CAS  Google Scholar 

  30. Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    CAS  PubMed  Google Scholar 

  31. Gregory, R. I. et al. The microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    CAS  PubMed  Google Scholar 

  32. Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. & Hannon, G. J. Processing of primary microRNAs by the microprocessor complex. Nature 432, 231–235 (2004).

    CAS  PubMed  Google Scholar 

  33. Han, J. et al. The Drosha–DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006). Elegant study showing the molecular mechanism of the first step involved in pri-miRNA processing.

    CAS  PubMed  Google Scholar 

  35. Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    CAS  PubMed  Google Scholar 

  36. Bohnsack, M. T., Czaplinski, K. & Gorlich, D. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. Rna 10, 185–191 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yi, R., Qin, Y., Macara, I. G. & Cullen, B. R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Chu, C. & Rana, T. M. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 4, e210 (2006).

    PubMed  PubMed Central  Google Scholar 

  39. Sen, G. L. & Blau, H. M. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nature Cell Biol. 7, 633–636 (2005).

    CAS  PubMed  Google Scholar 

  40. Pillai, R. S. et al. Inhibition of translational initiation by let-7 microRNA in human cells. Science 309, 1573–1576 (2005).

    CAS  PubMed  Google Scholar 

  41. Liu, J., Valencia-Sanchez, M. A., Hannon, G. J. & Parker, R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nature Cell Biol. 7, 719–723 (2005). This paper, combined with references 38–40, showed that Argonaute proteins localize to mRNA-processing bodies, or P-bodies.

    CAS  PubMed  Google Scholar 

  42. Valencia-Sanchez, M. A., Liu, J., Hannon, G. J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006).

    CAS  PubMed  Google Scholar 

  43. Kedersha, N. et al. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169, 871–884 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Chiu, Y. L. & Rana, T. M. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561 (2002).

    CAS  PubMed  Google Scholar 

  45. Chiu, Y. L. & Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034–1048 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Haley, B. & Zamore, P. D. Kinetic analysis of the RNAi enzyme complex. Nature Struct. Mol. Biol. 11, 599–606 (2004). This article, together with references 44 and 45, showed that RNA helical geometry is the major determinant in RNAi.

    CAS  Google Scholar 

  47. Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 31, 589–595 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Neenhold, H. R. & Rana, T. M. Major groove opening at the HIV-1 Tat binding site of TAR RNA evidenced by a rhodium probe. Biochemistry 34, 6303–6309 (1995).

    CAS  PubMed  Google Scholar 

  49. Weeks, K. M. & Crothers, D. M. Major groove accessibility of RNA. Science 261, 1574–1577 (1993).

    CAS  PubMed  Google Scholar 

  50. Holen, T., Amarzguioui, M., Babaie, E. & Prydz, H. Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway. Nucleic Acids Res. 31, 2401–2407 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Yekta, S., Shih, I. H. & Bartel, D. P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004).

    CAS  PubMed  Google Scholar 

  52. Hutvagner, G. & Zamore, P. D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002).

    CAS  PubMed  Google Scholar 

  53. Saxena, S., Jonsson, Z. O. & Dutta, A. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278, 44312–44319 (2003).

    CAS  PubMed  Google Scholar 

  54. Mello, C. C. & Conte, D. Revealing the world of RNA interference. Nature 431, 338–342 (2004).

    CAS  PubMed  Google Scholar 

  55. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399, 166–169 (1999).

    CAS  PubMed  Google Scholar 

  56. Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543–553 (2000).

    CAS  PubMed  Google Scholar 

  57. Lipardi, C., Wei, Q. & Paterson, B. M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001).

    CAS  PubMed  Google Scholar 

  58. Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533–542 (2000).

    CAS  PubMed  Google Scholar 

  59. Sijen, T. et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476 (2001).

    CAS  PubMed  Google Scholar 

  60. Lipardi, C., Wei, Q. & Paterson, B. M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001).

    CAS  PubMed  Google Scholar 

  61. Nishikura, K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107, 415–418 (2001).

    CAS  PubMed  Google Scholar 

  62. Czauderna, F. et al. Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 31, 2705–2716 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Schwarz, D. S., Hutvagner, G., Haley, B. & Zamore, P. D. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell 10, 537–548 (2002).

    CAS  PubMed  Google Scholar 

  64. Roignant, J. Y. et al. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. Rna 9, 299–308 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Ma, J. B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Parker, J. S., Roe, S. M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Jackson, A. L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. Rna 12, 1179–1187 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003).

    CAS  Google Scholar 

  69. Zimmermann, T. S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006).

    CAS  PubMed  Google Scholar 

  70. Morrissey, D. V. et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nature Biotechnol. 23, 1002–1007 (2005).

    CAS  Google Scholar 

  71. Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).

    CAS  PubMed  Google Scholar 

  72. Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. Rna 12, 1197–1205 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Filipowicz, W. RNAi: the nuts and bolts of the RISC machine. Cell 122, 17–20 (2005).

    CAS  PubMed  Google Scholar 

  74. Zamore, P. D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005).

    CAS  PubMed  Google Scholar 

  75. Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    CAS  PubMed  Google Scholar 

  76. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    CAS  PubMed  Google Scholar 

  77. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    CAS  PubMed  Google Scholar 

  78. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J. 20, 6877–6888 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  PubMed  Google Scholar 

  80. Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003). This paper, together with reference 79, showed the influence of siRNA thermodynamic stability on RISC formation.

    CAS  PubMed  Google Scholar 

  81. Leuschner, P. J., Ameres, S. L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & Zamore, P. D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).

    CAS  PubMed  Google Scholar 

  83. Rand, T. A., Petersen, S., Du, F. & Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005).

    CAS  PubMed  Google Scholar 

  84. Amarzguioui, M. & Prydz, H. An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun. 316, 1050–1058 (2004).

    CAS  PubMed  Google Scholar 

  85. Hohjoh, H. Enhancement of RNAi activity by improved siRNA duplexes. FEBS Lett. 557, 193–198 (2004).

    CAS  PubMed  Google Scholar 

  86. Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S. & Saigo, K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32, W124–W129 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Reynolds, A. et al. Rational siRNA design for RNA interference. Nature Biotechnol. 22, 326–330 (2004).

    CAS  Google Scholar 

  88. Ui-Tei, K. et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32, 936–948 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Yiu, S. M. et al. Filtering of ineffective siRNAs and improved siRNA design yool. Bioinformatics 21, 144–151 (2005).

    CAS  PubMed  Google Scholar 

  90. Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Sontheimer, E. J. Assembly and function of RNA silencing complexes. Nature Rev. Mol. Cell Biol. 6, 127–138 (2005).

    CAS  Google Scholar 

  92. Brown, K. M., Chu, C. Y. & Rana, T. M. Target accessibility dictates the potency of human RISC. Nature Struct. Mol. Biol. 12, 469–470 (2005).

    CAS  Google Scholar 

  93. Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. Rna 10, 544–550 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Chiu, Y. L., Cao, H., Jacque, J. M., Stevenson, M. & Rana, T. M. Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1). J. Virol. 78, 2517–2529 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Forstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3, e236 (2005).

    PubMed  PubMed Central  Google Scholar 

  97. Haase, A. D. et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 6, 961–967 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Caudy, A. A., Myers, M., Hannon, G. J. & Hammond, S. M. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Doench, J. G. & Sharp, P. A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Lim, L. P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    CAS  PubMed  Google Scholar 

  101. Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Schwarz, D. S., Tomari, Y. & Zamore, P. D. The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr. Biol. 14, 787–791 (2004).

    CAS  PubMed  Google Scholar 

  103. Schubert, S., Grunweller, A., Erdmann, V. A. & Kurreck, J. Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348, 883–893 (2005).

    CAS  PubMed  Google Scholar 

  104. Overhoff, M. et al. Local RNA target structure influences siRNA efficacy: a systematic global analysis. J. Mol. Biol. 348, 871–881 (2005). This article, together with references 92 and 103, showed the effect of target accessibility on RNAi function.

    CAS  PubMed  Google Scholar 

  105. Rana, T. M. & Jeang, K. T. Biochemical and functional interactions between HIV-1 Tat protein and TAR RNA. Arch. Biochem. Biophys. 365, 175–185 (1999).

    CAS  PubMed  Google Scholar 

  106. Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208 (1999).

    CAS  PubMed  Google Scholar 

  107. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    PubMed  Google Scholar 

  108. Dykxhoorn, D. M. & Lieberman, J. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu. Rev. Med. 56, 401–423 (2005).

    CAS  PubMed  Google Scholar 

  109. Palliser, D. et al. An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 439, 89–94 (2006).

    CAS  PubMed  Google Scholar 

  110. Vazquez, F. et al. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79 (2004).

    CAS  PubMed  Google Scholar 

  111. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. & Poethig, R. S. SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002).

    CAS  PubMed  Google Scholar 

  113. Reinhart, B. J. & Bartel, D. P. Small RNAs correspond to centromere heterochromatic repeats. Science 297, 1831 (2002).

    CAS  PubMed  Google Scholar 

  114. Aravin, A. A. et al. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr. Biol. 11, 1017–1027 (2001).

    CAS  PubMed  Google Scholar 

  115. Mochizuki, K. & Gorovsky, M. A. Small RNAs in genome rearrangement in Tetrahymena. Curr. Opin. Genet. Dev. 14, 181–187 (2004).

    CAS  PubMed  Google Scholar 

  116. Aravin, A. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 (2006).

    CAS  PubMed  Google Scholar 

  117. Girard, A., Sachidanandam, R., Hannon, G. J. & Carmell, M. A. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006).

    PubMed  Google Scholar 

  118. Grivna, S. T., Beyret, E., Wang, Z. & Lin, H. A novel class of small RNAs in mouse spermatogenic cells. Genes Dev. 20, 1709–1714 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Watanabe, T. et al. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev. 20, 1732–1743 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Lau, N. C. et al. Characterization of the piRNA complex from rat testes. Science 313, 363–367 (2006).

    CAS  PubMed  Google Scholar 

  121. Kuramochi-Miyagawa, S. et al. Two mouse piwi-related genes: miwi and mili. Mech. Dev. 108, 121–133 (2001).

    CAS  PubMed  Google Scholar 

  122. Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004).

    CAS  PubMed  Google Scholar 

  123. Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E. & Filipowicz, W. Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68 (2004).

    CAS  PubMed  Google Scholar 

  124. Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M. C. Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19, 2837–2848 (2005). This paper, together with references 75 and 76, provided evidence that AGO2 is the catalytic enzyme that cleaves target mRNA during RNAi.

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol. 3, e235 (2005).

    PubMed  PubMed Central  Google Scholar 

  126. Jiang, F. et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev. 19, 1674–1679 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Lee, Y. et al. The role of PACT in the RNA silencing pathway. Embo J. 25, 522–532 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Parker, J. S., Roe, S. M. & Barford, D. Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. Embo J. 23, 4727–4737 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Schubert, S. et al. Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs. J. Mol. Biol. 346, 457–465 (2005).

    CAS  PubMed  Google Scholar 

  130. Holen, T., Amarzguioui, M., Wiiger, M. T., Babaie, E. & Prydz, H. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res. 30, 1757–1766 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Prakash, T. P. et al. Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J. Med. Chem. 48, 4247–4253 (2005).

    CAS  PubMed  Google Scholar 

  132. Braasch, D. A. et al. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42, 7967–7975 (2003).

    CAS  PubMed  Google Scholar 

  133. Kraynack, B. A. & Baker, B. F. Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity. Rna 12, 163–176 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. Rivas, F. V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct. Mol. Biol. 12, 340–349 (2005).

    CAS  Google Scholar 

  135. Wu, H., Lima, W. F. & Crooke, S. T. Investigating the structure of human RNase H1 by site-directed mutagenesis. J. Biol. Chem. 276, 23547–23553 (2001).

    CAS  PubMed  Google Scholar 

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Acknowledgements

I am grateful to D. Barford, J.B. Ma and D. Patel for providing high-resolution RNA and RNA–protein structures and to members of my laboratory, especially C. Chu for her assistance with illustrations and H. Cao for kinetic data analysis. This work was supported in part by a grant from the National Institutes of Health to T.M.R.

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Glossary

P-body

A cytoplasmic structure that is involved in storing and degrading translationally repressed RNA.

Ribonucleoprotein particle

(RNP). A complex of proteins and RNA.

A-form helix

A right-handed (clockwise) helix in which base pairs are significantly tilted with respect to the central axis of the helix. The grooves are not as well defined as in the B-form helix. RNA commonly adopts an A-form helical configuration.

DEAD-box RNA-helicase domain

An evolutionarily conserved domain in a family of enzymes that use ATP hydrolysis to unwind RNA duplexes. The domain is named after the DEAD (Asp-Glu-Ala-Asp) motif.

RIII domain

A conserved domain that is present in the RNase III-type enzymes Dicer and Drosha and that is involved in endonuclease reactions that result in cleaving double-stranded RNA substrates.

dsRNA-binding domain (dsRBD)

A conserved protein region that binds double-stranded RNA.

PAZ domain

A conserved domain that is found in Argonaute- and Dicer-family proteins and that specifically binds to small RNA helices.

B-form helix

The Watson–Crick double helix of DNA, a right-handed (clockwise) helix in which 10 base pairs complete a single 360° rotation (helical turn). Grooves are prominent and well defined.

Sugar puckers

Ribose and deoxyribose sugar rings in RNA and DNA, respectively, are made of five atoms (one oxygen and four carbons (C1′–4′)), one of which lies out of the plane of the others. These sugar rings are flexible and dynamic structures that twist C2′ or C3′ atoms out of the plane, resulting in twisted forms or puckers.

Endo configuration

A sugar-ring configuration in which a specific carbon atom (C2′ or C3′) twists up from the plane with respect to the other four atoms of the ring.

Helical pitch

The length of one complete helical turn.

Off-target effects

In the context of RNA silencing, this refers to the decreased expression of genes other than the intended target gene.

Argonaute-family proteins

A group of proteins that are characterized by the presence of two conserved domains, PAZ and PIWI. These proteins are essential for diverse RNA-silencing pathways.

PIWI domain

A conserved domain that is found in Argonaute-family proteins and that has structural similarities to RNase H. At least in some cases, it cleaves the RNA strand of an RNA–DNA helix.

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Rana, T. Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8, 23–36 (2007). https://doi.org/10.1038/nrm2085

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