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:

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans

Nature Structural & Molecular Biology volume 16, pages 207–211 (2009)Cite this article

Abstract

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.

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: Mismatched siRNA duplexes bypass mutations in the RDE-1 DDH motif.
Figure 2: In RDE-1 DDH mutants, the passenger strand accumulates and remains associated with the guide strand and RDE-1.
Figure 3: Passenger-strand turnover is required for target recognition.

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. 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 

  3. 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 

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

    Article  CAS  Google Scholar 

  5. Peters, L. & Meister, G. Argonaute proteins: mediators of RNA silencing. Mol. Cell 26, 611–623 (2007).

    Article  CAS  Google Scholar 

  6. Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426, 465–469 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Yan, K.S. et al. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474 (2003).

    Article  Google Scholar 

  12. Baumberger, N. & Baulcombe, D.C. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA 102, 11928–11933 (2005).

    Article  CAS  Google Scholar 

  13. Gunawardane, L.S. et al. A Slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Saito, K. et al. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20, 2214–2222 (2006).

    Article  CAS  Google Scholar 

  16. Irvine, D.V. et al. Argonaute slicing is required for heterochromatic silencing and spreading. Science 313, 1134–1137 (2006).

    Article  CAS  Google Scholar 

  17. 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 

  18. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. & Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002).

    Article  CAS  Google Scholar 

  21. 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 

  22. Buker, S.M. et al. Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast. Nat. Struct. Mol. Biol. 14, 200–207 (2007).

    Article  CAS  Google Scholar 

  23. Maiti, M., Lee, H.C. & Liu, Y. QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes Dev. 21, 590–600 (2007).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Kim, K., Lee, Y.S. & Carthew, R.W. Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes. RNA 13, 22–29 (2007).

    Article  CAS  Google Scholar 

  28. 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 

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

    Article  CAS  Google Scholar 

  30. 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 

  31. 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 

  32. Aoki, K., Moriguchi, H., Yoshioka, T., Okawa, K. & Tabara, H. In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans. EMBO J. 26, 5007–5019 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. 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 

  36. 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 

  37. Förstemann, 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 

Download references

Acknowledgements

We thank C. Mello (University of Massachusetts Medical School) for providing strains and B. Ason, T. Sixma and M. Bühler for help and discussions. The work was supported by a VIDI fellowship from the Dutch Scientific Organization (NWO) to T.S. and the Sixth Framework Programme of the European Commission through the SIROCCO Integrated Project to R.F.K.

Author information

Author notes

  1. Titia Sijen

    Present address: Present address: Netherlands Forensic Institute, P.O. Box 24044, 2490 AA, The Hague, The Netherlands.,

  2. Titia Sijen and René F Ketting: These authors contributed equally to this work.

Authors and Affiliations

  1. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

    Florian A Steiner, Kristy L Okihara, Suzanne W Hoogstrate, Titia Sijen & René F Ketting

Authors
  1. Florian A Steiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristy L Okihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzanne W Hoogstrate
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Titia Sijen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. René F Ketting
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.A.S., T.S. and R.F.K. designed the experiments; F.A.S., K.L.O., S.W.H. and T.S. performed the experiments; F.A.S. and R.F.K. wrote the paper.

Corresponding author

Correspondence to René F Ketting.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 629 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Steiner, F., Okihara, K., Hoogstrate, S. et al. RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans. Nat Struct Mol Biol 16, 207–211 (2009). https://doi.org/10.1038/nsmb.1541

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1541

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