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.

  • Article
  • Published:

Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

Nature Structural & Molecular Biology volume 14, pages 927–933 (2007)Cite this article

Abstract

In C. elegans, DCR-1 is required for the maturation of both short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are subsequently loaded into different Argonaute proteins to mediate silencing via distinct mechanisms. We used in vivo analyses to show that precursors of small RNAs contain structural features that direct the small RNAs into the RNA interference (RNAi) pathway or the miRNA-processing pathway. Nucleotide changes in the pre-let-7 miRNA precursor that make its stem fully complementary cause the resulting small RNA to be recognized as siRNA and induce binding to RDE-1, which leads to RNAi. Mismatches of 1 to 3 nucleotides at various positions in the stem of the precursor restore direction into the miRNA pathway, as the largest portion of such small RNA variants is associated with ALG-1. The Argonaute proteins to which the small RNAs are bound determine the silencing mode, and no functional overlap between RDE-1 and ALG-1 was detected.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Characteristics of pre-let-7 and the transgene-derived pre-miRNAs and pre-siRNAs used in this study.
Figure 2: Small RNAs derived from mismatched precursors have an miRNA-like distribution in size fractions and fractionate with HA–ALG-1.
Figure 3: Small RNAs derived from mismatched precursors are bound by ALG-1 rather than RDE-1.
Figure 4: RDE-1 is associated mainly with small RNAs derived from fully matching precursors.

Similar content being viewed by others

References

  1. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Kim, V.N. Small RNAs: classification, biogenesis, and function. Mol. Cells 19, 1–15 (2005).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Zhang, H., Kolb, F.A., Brondani, V., Billy, E. & Filipowicz, W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885 (2002).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Lee, Y., Jeon, K., Lee, J.T., Kim, S. & Kim, V.N. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21, 4663–4670 (2002).

    Article  CAS  Google Scholar 

  11. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104 (2004).

    Article  Google Scholar 

  16. Vazquez, F. Arabidopsis endogenous small RNAs: highways and byways. Trends Plant Sci. 11, 460–468 (2006).

    Article  CAS  Google Scholar 

  17. Brodersen, P. & Voinnet, O. The diversity of RNA silencing pathways in plants. Trends Genet. 22, 268–280 (2006).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Pillai, R.S., Bhattacharyya, S.N. & Filipowicz, W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 17, 118–126 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Caudy, A.A. & Hannon, G.J. Induction and biochemical purification of RNA-induced silencing complex from Drosophila S2 cells. Methods Mol. Biol. 265, 59–72 (2004).

    CAS  PubMed  Google Scholar 

  28. Rand, T.A., Ginalski, K., Grishin, N.V. & Wang, X. Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity. Proc. Natl. Acad. Sci. USA 101, 14385–14389 (2004).

    Article  CAS  Google Scholar 

  29. 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).

    Article  Google Scholar 

  30. 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).

    Article  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Tabara, H., Yigit, E., Siomi, H. & Mello, C.C. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871 (2002).

    Article  CAS  Google Scholar 

  33. Parker, G.S., Eckert, D.M. & Bass, B.L. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA 12, 807–818 (2006).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Tops, B.B., Plasterk, R.H. & Ketting, R.F. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: almost identical yet different. Cold Spring Harb. Symp. Quant. Biol. 71, 189–194 (2006).

    Article  CAS  Google Scholar 

  37. Caudy, A.A. et al. A micrococcal nuclease homologue in RNAi effector complexes. Nature 425, 411–414 (2003).

    Article  CAS  Google Scholar 

  38. Lu, R. et al. Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature 436, 1040–1043 (2005).

    Article  CAS  Google Scholar 

  39. Wilkins, C. et al. RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature 436, 1044–1047 (2005).

    Article  CAS  Google Scholar 

  40. Plasterk, R.H. RNA silencing: the genome's immune system. Science 296, 1263–1265 (2002).

    Article  CAS  Google Scholar 

  41. Tomari, Y., Du, T. & Zamore, P.D. Sorting of Drosophila small silencing RNAs. Cell 130, 299–308 (2007).

    Article  CAS  Google Scholar 

  42. 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).

    Article  Google Scholar 

  43. Lee, R.C., Hammell, C.M. & Ambros, V. Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA 12, 589–597 (2006).

    Article  CAS  Google Scholar 

  44. Duchaine, T.F. et al. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell 124, 343–354 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Ruby, J.G. et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Hannon and F. Rivas (Cold Spring Harbor Laboratory) for providing reagents and help with the capture assays, B. Tops (Hubrecht Institute) and C. Mello (University of Massachusetts Medical School) for providing strains and W. Kloosterman for discussions and critical reading of the manuscript. This work was supported by a Vidi fellowship from the Dutch Scientific Organization (NWO) to T.S. and a European Union grant (HPRN-CT-2002-00257) and Netherlands Genomics Initiative grant (050-72-415) to F.A.S.

Author information

Author notes

  1. Karen L Thijssen & Titia Sijen

    Present address: Present addresses: University Medical Center Groningen, Groningen, 9700 RB, The Netherlands (K.L.T.) and Netherlands Forensic Institute, The Hague, 2497 GB, The Netherlands (T.S.).,

Authors and Affiliations

  1. Hubrecht Institute, Koninklijke Nederlandse Akademie van Wetenschappen, Utrecht, 3584 CT, The Netherlands

    Florian A Steiner, Suzanne W Hoogstrate, Kristy L Okihara, Karen L Thijssen, Rene F Ketting, Ronald H A Plasterk & Titia Sijen

Authors
  1. Florian A Steiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suzanne W Hoogstrate
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristy L Okihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karen L Thijssen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rene F Ketting
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ronald H A Plasterk
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Titia Sijen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.A.S., S.W.H., K.L.O., K.L.T. and T.S. carried out experiments; T.S., F.A.S., R.F.K. and R.H.A.P. supervised the research; F.A.S. and T.S. wrote the paper.

Corresponding authors

Correspondence to Florian A Steiner or Titia Sijen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1677 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Steiner, F., Hoogstrate, S., Okihara, K. et al. Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans. Nat Struct Mol Biol 14, 927–933 (2007). https://doi.org/10.1038/nsmb1308

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1308

This article is cited by

Search

Advanced 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