Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Small RNA sorting: matchmaking for Argonautes

Key Points

  • Small RNAs are defined by their size (~20–30 nucleotides in length) and their association with members of the Argonaute family. They impact nearly every biological process in eukaryotic cells, directly or indirectly.

  • MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are born from dsRNA precursors, whereas Piwi-interacting RNAs (piRNAs) originate from single-stranded transcripts.

  • To perform their myriad roles, different classes of small RNAs must not only be generated in a precise manner, but must also be sorted into specific Argonaute complexes. An Argonaute protein primed with a single-stranded small RNA is called an RNA-induced silencing complex (RISC).

  • Eukaryotic organisms often encode several Argonaute proteins that function in distinct pathways. They typically show various preferences for the small RNAs they accept, comprising loading determinants that include the identity of terminal nucleotides, small RNA duplex structure and thermodynamic properties.

  • Small RNA duplexes are usually not incorporated into Argonaute proteins without assistance from additional protein factors, known as the RISC-loading machinery.

  • miRNAs and siRNAs arise from small RNA duplexes and are loaded into Argonaute as dsRNA molecules. Thus, during RISC maturation, one strand must be selected specifically, whereas the other strand must be lost or degraded.

  • Mature RISC regulates targets through sequence complementarity. The ultimate impact of accurate strand selection and sorting is that an active RISC is formed, imbued with the ability to regulate target transcripts.

  • Biogenesis and sorting of small RNAs in animals and plants share some key mechanistic features, but have also evolved myriad variations and adaptations.

Abstract

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: MicroRNA biogenesis in Drosophila melanogaster.
Figure 2: Production of small interfering RNAs.
Figure 3: Small RNA sorting and RNA-induced silencing complex assembly in flies.

Similar content being viewed by others

References

  1. Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Rev. Genet. 10, 94–108 (2009).

    CAS  PubMed  Google Scholar 

  2. Chapman, E. J. & Carrington, J. C. Specialization and evolution of endogenous small RNA pathways. Nature Rev. Genet. 8, 884–896 (2007).

    CAS  PubMed  Google Scholar 

  3. Voinnet, O. Origin, biogenesis, and activity of plant microRNAs. Cell 136, 669–687 (2009).

    CAS  PubMed  Google Scholar 

  4. Vagin, V. V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324 (2006).

    CAS  PubMed  Google Scholar 

  5. Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Pak, J. & Fire, A. Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 315, 241–244 (2007).

    CAS  PubMed  Google Scholar 

  7. Sijen, T., Steiner, F. A., Thijssen, K. L. & Plasterk, R. H. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315, 244–247 (2007).

    CAS  PubMed  Google Scholar 

  8. Houwing, S. et al. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell 129, 69–82 (2007).

    CAS  PubMed  Google Scholar 

  9. Yigit, E. et al. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127, 747–757 (2006).

    CAS  PubMed  Google Scholar 

  10. Tolia, N. H. & Joshua-Tor, L. Slicer and the Argonautes. Nature Chem. Biol. 3, 36–43 (2007).

    CAS  Google Scholar 

  11. Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008).

    CAS  Google Scholar 

  12. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 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 

  14. 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). References 12–14 described passenger strand cleavage and showed that Argonaute proteins themselves function in RISC maturation.

    CAS  PubMed  Google Scholar 

  15. Fabian, M. R., Sonenberg, N. & Filipowicz, W. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem. 79, 351–379 (2010).

    CAS  PubMed  Google Scholar 

  16. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Brodersen, P. & Voinnet, O. Revisiting the principles of microRNA target recognition and mode of action. Nature Rev. Mol. Cell Biol. 10, 141–148 (2009).

    CAS  Google Scholar 

  18. Rodriguez, A., Griffiths-Jones, S., Ashurst, J. L. & Bradley, A. Identification of mammalian microRNA host genes and transcription units. Genome Res. 14, 1902–1910 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, Y. et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Cai, X., Hagedorn, C. H. & Cullen, B. R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 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 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 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 

  25. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006).

    CAS  PubMed  Google Scholar 

  26. 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 

  27. 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 

  28. 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 

  29. Bussing, I., Yang, J. S., Lai, E. C. & Grosshans, H. The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila. EMBO J. 29, 1830–1839 (2010).

    PubMed  PubMed Central  Google Scholar 

  30. Gruber, J. J. et al. Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation. Cell 138, 328–339 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Sabin, L. R. et al. Ars2 regulates both miRNA- and siRNA-dependent silencing and suppresses RNA virus infection in Drosophila. Cell 138, 340–351 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 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). This paper identified the ribonuclease Dicer as the dsRNA processing enzyme.

    CAS  PubMed  Google Scholar 

  33. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001).

    CAS  PubMed  Google Scholar 

  34. Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001).

    CAS  PubMed  Google Scholar 

  35. Ketting, R. F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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 

  37. 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 

  38. 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 

  39. 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 

  40. 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 

  41. Park, J. K., Liu, X., Strauss, T. J., McKearin, D. M. & Liu, Q. The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells. Curr. Biol. 17, 533–538 (2007).

    CAS  PubMed  Google Scholar 

  42. Zhou, R. et al. Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform. RNA 15, 1886–1895 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Berezikov, E., Chung, W. J., Willis, J., Cuppen, E. & Lai, E. C. Mammalian mirtron genes. Mol. Cell 28, 328–336 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Okamura, K., Hagen, J. W., Duan, H., Tyler, D. M. & Lai, E. C. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130, 89–100 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ruby, J. G., Jan, C. H. & Bartel, D. P. Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83–86 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Flynt, A. S., Greimann, J. C., Chung, W. J., Lima, C. D. & Lai, E. C. MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila. Mol. Cell 38, 900–907 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Yu, B. et al. The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc. Natl Acad. Sci. USA 105, 10073–10078 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B. & Bartel, D. P. MicroRNAs in plants. Genes Dev. 16, 1616–1626 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Park, W., Li, J., Song, R., Messing, J. & Chen, X. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12, 1484–1495 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nature Genet. 38, 721–725 (2006).

    CAS  PubMed  Google Scholar 

  51. Kurihara, Y. & Watanabe, Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl Acad. Sci. USA 101, 12753–12758 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Han, M. H., Goud, S., Song, L. & Fedoroff, N. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl Acad. Sci. USA 101, 1093–1098 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Kurihara, Y., Takashi, Y. & Watanabe, Y. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–212 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Vazquez, F., Gasciolli, V., Crete, P. & Vaucheret, H. The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14, 346–351 (2004).

    CAS  PubMed  Google Scholar 

  55. Dong, Z., Han, M. H. & Fedoroff, N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl Acad. Sci. USA 105, 9970–9975 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Lobbes, D., Rallapalli, G., Schmidt, D. D., Martin, C. & Clarke, J. SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7, 1052–1058 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Yang, L., Liu, Z., Lu, F., Dong, A. & Huang, H. SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J. 47, 841–850 (2006).

    CAS  PubMed  Google Scholar 

  58. Laubinger, S. et al. Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 105, 8795–8800 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Gregory, B. D. et al. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14, 854–866 (2008).

    CAS  PubMed  Google Scholar 

  60. Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr. Biol. 15, 1501–1507 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Yang, Z., Ebright, Y. W., Yu, B. & Chen, X. HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res. 34, 667–675 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Yu, B. et al. Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Ramachandran, V. & Chen, X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis. Science 321, 1490–1492 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Ameres, S. L. et al. Target RNA-directed trimming and tailing of small silencing RNAs. Science 328, 1534–1539 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H. & Poethig, R. S. Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl Acad. Sci. USA 102, 3691–3696 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Eamens, A. L., Smith, N. A., Curtin, S. J., Wang, M. B. & Waterhouse, P. M. The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15, 2219–2235 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999). This seminal report was the first to link small RNAs to post-transcriptional gene silencing.

    CAS  PubMed  Google Scholar 

  68. 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 

  69. Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003).

    CAS  PubMed  Google Scholar 

  70. Hartig, J. V., Esslinger, S., Bottcher, R., Saito, K. & Forstemann, K. Endo-siRNAs depend on a new isoform of Loquacious and target artificially introduced, high-copy sequences. EMBO J. 28, 2932–2944 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Miyoshi, K., Miyoshi, T., Hartig, J. V., Siomi, H. & Siomi, M. C. Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila. RNA 16, 506–515 (2010).

    PubMed  PubMed Central  Google Scholar 

  72. Marques, J. T. et al. Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila. Nature Struct. Mol. Biol. 17, 24–30 (2010).

    CAS  Google Scholar 

  73. Ghildiyal, M. et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320, 1077–1081 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803–806 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Kawamura, Y. et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793–797 (2008).

    CAS  PubMed  Google Scholar 

  76. Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798–802 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Chung, W. J., Okamura, K., Martin, R. & Lai, E. C. Endogenous RNA interference provides a somatic defense against Drosophila transposons. Curr. Biol. 18, 795–802 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Lipardi, C. & Paterson, B. M. Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression. Proc. Natl Acad. Sci. USA 106, 15645–15650 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).

    CAS  PubMed  Google Scholar 

  81. Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P. & Blelloch, R. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev. 22, 2773–2785 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Knight, S. W. & Bass, B. L. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269–2271 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Steiner, F. A., Okihara, K. L., Hoogstrate, S. W., Sijen, T. & Ketting, R. F. RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans. Nature Struct. Mol. Biol. 16, 207–211 (2009).

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  85. 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 

  86. Yoshikawa, M., Peragine, A., Park, M. Y. & Poethig, R. S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Williams, L., Carles, C. C., Osmont, K. S. & Fletcher, J. C. A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes. Proc. Natl Acad. Sci. USA 102, 9703–9708 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005).

    CAS  PubMed  Google Scholar 

  89. Axtell, M. J., Jan, C., Rajagopalan, R. & Bartel, D. P. A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565–577 (2006).

    CAS  PubMed  Google Scholar 

  90. Montgomery, T. A. et al. Specificity of ARGONAUTE7–miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 (2008).

    CAS  PubMed  Google Scholar 

  91. Hernandez-Pinzon, I. et al. SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA. Plant J. 50, 140–148 (2007).

    CAS  PubMed  Google Scholar 

  92. Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R. & Zhu, J. K. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123, 1279–1291 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Katiyar-Agarwal, S. et al. A pathogen-inducible endogenous siRNA in plant immunity. Proc. Natl Acad. Sci. USA 103, 18002–18007 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Henz, S. R. et al. Distinct expression patterns of natural antisense transcripts in Arabidopsis. Plant Physiol. 144, 1247–1255 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, e104 (2004).

    PubMed  PubMed Central  Google Scholar 

  96. Chan, S. W. et al. RNA silencing genes control de novo DNA methylation. Science 303, 1336 (2004).

    CAS  PubMed  Google Scholar 

  97. Onodera, Y. et al. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120, 613–622 (2005).

    CAS  PubMed  Google Scholar 

  98. Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).

    CAS  PubMed  Google Scholar 

  99. Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 19, 2030–2040 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Kasschau, K. D. et al. Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol. 5, e57 (2007).

    PubMed  PubMed Central  Google Scholar 

  101. Smith, L. M. et al. An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 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 

  103. Maniataki, E. & Mourelatos, Z. A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–2990 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  PubMed  Google Scholar 

  105. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003). References 104 and 105 identified intrinsic determinants in small RNA duplexes that affect their sorting into Argonaute complexes.

    CAS  PubMed  Google Scholar 

  106. Ghildiyal, M., Xu, J., Seitz, H., Weng, Z. & Zamore, P. D. Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. RNA 16, 43–56 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Czech, B. et al. Hierarchical rules for Argonaute loading in Drosophila. Mol. Cell 36, 445–456 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. 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 

  109. Hu, H. Y. et al. Sequence features associated with microRNA strand selection in humans and flies. BMC Genomics 10, 413 (2009).

    PubMed  PubMed Central  Google Scholar 

  110. Okamura, K., Liu, N. & Lai, E. C. Distinct mechanisms for microRNA strand selection by Drosophila Argonautes. Mol. Cell 36, 431–444 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010). This recent study found structural evidence in Argonaute proteins that explains 5′ terminal nucleotide biases.

    CAS  PubMed  Google Scholar 

  112. Mi, S. et al. Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008). Together with reference 90, this paper described the identification of 5′ terminal nucleotides as sorting determinants in plants.

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Forstemann, K., Horwich, M. D., Wee, L., Tomari, Y. & Zamore, P. D. Drosophila microRNAs are sorted into functionally distinct Argonaute complexes after production by Dicer-1. Cell 130, 287–297 (2007).

    PubMed  PubMed Central  Google Scholar 

  114. Ameres, S. L., Hung, J.-H., Xu, J., Weng, Z. & Zamore, P. D. Target RNA-directed tailing and trimming purifies the sorting of endo-siRNAs between the two Drosophila Argonaute proteins. RNA (in the press).

  115. Steiner, F. A. et al. Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans. Nature Struct. Mol. Biol. 14, 927–933 (2007).

    CAS  Google Scholar 

  116. Jannot, G., Boisvert, M. E., Banville, I. H. & Simard, M. J. Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans. RNA 14, 829–835 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  118. Yoda, M. et al. ATP-dependent human RISC assembly pathways. Nature Struct. Mol. Biol. 17, 17–23 (2010).

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  120. Wu, L. et al. Rice microRNA effector complexes and targets. Plant Cell 21, 3421–3435 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M. & Watanabe, Y. The mechanism selecting the guide strand from small RNA duplexes is different among Argonaute proteins. Plant Cell Physiol. 49, 493–500 (2008).

    CAS  PubMed  Google Scholar 

  122. Yang, J. S. et al. Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis. Proc. Natl Acad. Sci. USA 107, 15163–15168 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A Dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Cifuentes, D. et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328, 1694–1698 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Diederichs, S. & Haber, D. A. Dual role for Argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell 131, 1097–1108 (2007).

    CAS  PubMed  Google Scholar 

  126. Tan, G. S. et al. Expanded RNA-binding activities of mammalian Argonaute 2. Nucleic Acids Res. 37, 7533–7545 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Das, P. P. et al. Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline. Mol. Cell 31, 79–90 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Batista, P. J. et al. PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Mol. Cell 31, 67–78 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Nykanen, A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001).

    CAS  PubMed  Google Scholar 

  130. Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841 (2004).

    CAS  PubMed  Google Scholar 

  131. Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W. & Sontheimer, E. J. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94 (2004).

    CAS  PubMed  Google Scholar 

  132. Kawamata, T., Seitz, H. & Tomari, Y. Structural determinants of miRNAs for RISC loading and slicer-independent unwinding. Nature Struct. Mol. Biol. 16, 953–960 (2009).

    CAS  Google Scholar 

  133. Johnston, M., Geoffroy, M. C., Sobala, A., Hay, R. & Hutvagner, G. HSP90 protein stabilizes unloaded argonaute complexes and microscopic P-bodies in human cells. Mol. Biol. Cell 21, 1462–1469 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. Iki, T. et al. In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol. Cell 39, 282–291 (2010).

    CAS  PubMed  Google Scholar 

  135. Iwasaki, S. et al. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol. Cell 39, 292–299 (2010).

    CAS  PubMed  Google Scholar 

  136. Miyoshi, T., Takeuchi, A., Siomi, H. & Siomi, M. C. A direct role for Hsp90 in pre-RISC formation in Drosophila. Nature Struct. Mol. Biol. 17, 1024–1026 (2010).

    CAS  Google Scholar 

  137. Tomari, Y., Du, T. & Zamore, P. D. Sorting of Drosophila small silencing RNAs. Cell 130, 299–308 (2007). References 113, 118, 132 and 137 made important contributions to understanding small RNA sorting and showed that miRNA maturation is independent of cleavage activity.

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Liu, X., Jiang, F., Kalidas, S., Smith, D. & Liu, Q. Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12, 1514–1520 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P. D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004).

    CAS  PubMed  Google Scholar 

  140. Liu, Y. et al. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325, 750–753 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Miyoshi, K., Okada, T. N., Siomi, H. & Siomi, M. C. Characterization of the miRNA–RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA 15, 1282–1291 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Horwich, M. D. et al. The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272 (2007).

    CAS  PubMed  Google Scholar 

  143. Saito, K., Sakaguchi, Y., Suzuki, T., Siomi, H. & Siomi, M. C. Pimet, the Drosophila homolog of HEN1, mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends. Genes Dev. 21, 1603–1608 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. Davis, E. et al. RNAi-mediated allelic trans-interaction at the imprinted Rtl1/Peg11 locus. Curr. Biol. 15, 743–749 (2005).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  146. Rhoades, M. W. et al. Prediction of plant microRNA targets. Cell 110, 513–520 (2002).

    CAS  PubMed  Google Scholar 

  147. Brodersen, P. et al. Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190 (2008).

    CAS  PubMed  Google Scholar 

  148. Wierzbicki, A. T., Ream, T. S., Haag, J. R. & Pikaard, C. S. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nature Genet. 41, 630–634 (2009).

    CAS  PubMed  Google Scholar 

  149. Matzke, M., Kanno, T., Daxinger, L., Huettel, B. & Matzke, A. J. RNA-mediated chromatin-based silencing in plants. Curr. Opin. Cell Biol. 21, 367–376 (2009).

    CAS  PubMed  Google Scholar 

  150. Chan, S. W., Henderson, I. R. & Jacobsen, S. E. Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Rev. Genet. 6, 351–360 (2005).

    CAS  PubMed  Google Scholar 

  151. Mosher, R. A., Schwach, F., Studholme, D. & Baulcombe, D. C. PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc. Natl Acad. Sci. USA 105, 3145–3150 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  152. Yin, H. & Lin, H. An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster. Nature 450, 304–308 (2007).

    CAS  PubMed  Google Scholar 

  153. Carmell, M. A. et al. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12, 503–514 (2007).

    CAS  PubMed  Google Scholar 

  154. Aravin, A. A. et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31, 785–799 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. Guang, S. et al. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 465, 1097–1101 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. Werner, S., Wollmann, H., Schneeberger, K. & Weigel, D. Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr. Biol. 20, 42–48 (2010).

    CAS  PubMed  Google Scholar 

  157. Mateos, J. L., Bologna, N. G., Chorostecki, U. & Palatnik, J. F. Identification of microRNA processing determinants by random mutagenesis of Arabidopsis MIR172a precursor. Curr. Biol. 20, 49–54 (2010).

    CAS  PubMed  Google Scholar 

  158. Song, L., Axtell, M. J. & Fedoroff, N. V. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr. Biol. 20, 37–41 (2010).

    CAS  PubMed  Google Scholar 

  159. Cuperus, J. T. et al. Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc. Natl Acad. Sci. USA 107, 466–471 (2010).

    CAS  PubMed  Google Scholar 

  160. Bologna, N. G., Mateos, J. L., Bresso, E. G. & Palatnik, J. F. A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J. 28, 3646–3656 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  161. Wang, Y. et al. Structure of an Argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  162. Song, J. J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nature Struct. Biol. 10, 1026–1032 (2003).

    CAS  PubMed  Google Scholar 

  163. Ma, J. B., Ye, K. & Patel, D. J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  164. 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 

  165. 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 

  166. Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing Argonaute silencing complex. Nature 456, 209–213 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  167. Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  168. 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 

  169. 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 

  170. Yuan, Y. R. et al. Crystal structure of A. aeolicus Argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  171. 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 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  173. Iwasaki, S., Kawamata, T. & Tomari, Y. Drosophila Argonaute1 and Argonaute2 employ distinct mechanisms for translational repression. Mol. Cell 34, 58–67 (2009).

    CAS  PubMed  Google Scholar 

  174. Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank O. Tam, F. Muerdter, J.-W. Wang and R. Zhou for comments on the manuscript. The authors are greatly indebted to J. Duffy for assistance with figures. B.C. is supported by a Ph.D fellowship from the Boehringer Ingelheim Fonds. This work was supported by grants from the National Institutes of Health and a kind gift from K. W. Davis. G.J.H. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gregory J. Hannon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

DATABASES

miRBase

mir-144

miR173

miR390

miR-451

FURTHER INFORMATION

Gregory J. Hannon's homepage

Gregory J. Hannon's Cold Spring Harbor Laboratory homepage

Glossary

RNase III protein

A member of a family of ribonucleases that process dsRNA, leaving 5′ monophosphates and 2-nt 3′ overhangs with hydroxyl ends. Drosha and Dicer are examples of such ribonucleases.

RNA-induced silencing complex

A regulatory multi-protein complex containing an Argonaute protein bound to a single-stranded small RNA that regulates gene expression through sequence complementarity between the guide RNA and the target transcript.

Guide strand

During RISC loading, one strand of an siRNA is selected and stabilized. This is termed the guide strand, and it confers target specificity. miRNA guide strands are termed miR strands.

Passenger strand

The non-incorporated strand of the siRNA duplex that is degraded during the assembly of RISC. Non-incorporated strands of miRNAs are called miR* strands.

Stem–loop structure

A region of dsRNA (stem) connected by an unpaired region (loop) in a single RNA molecule. This is a structure typical for miRNA precursors.

Mirtron

A miRNA that originates from a very short intron and is excised to form a pre-miRNA by the splicing machinery (and occasionally subsequent trimming), therefore bypassing the Drosha processing step.

Dicing

Refers to the cleavage events carried out by the RNase III family nuclease Dicer.

RNA-dependent RNA polymerase

An RNA polymerase that uses ssRNA as a template to synthesize dsRNA.

Trans-acting siRNA

A plant small RNA that primarily associates with AGO2. ta-siRNA biogenesis depends on miRNA-mediated cleavage of precursors that are further processed by DCL4 and other siRNA machinery factors.

Natural antisense transcript-derived siRNA

A stress-induced small RNA produced by DCL1 and DCL2 that originates from dsRNA formed by convergent transcription.

Heterochromatic siRNA

A highly abundant plant small RNA that arises from transposons and repeats. hc-siRNAs depend on DCL3 and mainly load into AGO4.

RNase H

A conserved family of endonucleases that cleave the RNA strand of RNA:DNA hybrid duplexes. AGO proteins contain RNase H-like domains.

Seed region

A region consisting of nucleotides 2–8 counted from the 5′ end of miRNAs that participates in the interaction between a small RNA and target transcript.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Czech, B., Hannon, G. Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 12, 19–31 (2011). https://doi.org/10.1038/nrg2916

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg2916

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing