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:

Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi

Nature Structural & Molecular Biology volume 19, pages 90–97 (2012)Cite this article

Subjects

An Erratum to this article was published on 05 February 2013

This article has been updated

Abstract

Endogenous RNA interference (endo-RNAi) pathways use a variety of mechanisms to generate siRNA and to mediate gene silencing. In Caenorhabditis elegans, DCR-1 is essential for competing RNAi pathways—the ERI endo-RNAi pathway and the exogenous RNAi pathway—to function. Here, we demonstrate that DCR-1 forms exclusive complexes in each pathway and further define the ERI–DCR-1 complex. We show that the tandem tudor protein ERI-5 potentiates ERI endo-RNAi by tethering an RNA-dependent RNA polymerase (RdRP) module to DCR-1. In the absence of ERI-5, the RdRP module is uncoupled from DCR-1. Notably, EKL-1, an ERI-5 paralog that specifies distinct RdRP modules in Dicer-independent endo-RNAi pathways, partially compensates for the loss of ERI-5 without interacting with DCR-1. Our results implicate tudor proteins in the recruitment of RdRP complexes to specific steps within DCR-1-dependent and DCR-1-independent endo-RNAi pathways.

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: Distinct DCR-1 complexes initiate endo- and exo-RNAi.
Figure 2: ERI-5 promotes the association of an RdRP module to the DCR-1 N terminus.
Figure 3: ERI-5 potentiates ERI endo-RNAi small RNA biogenesis.
Figure 4: Tandem tudor domain proteins are required for ERI endo-siRNA biogenesis.
Figure 5: Roles and paralog organization of RdRP modules in ERI endo-RNAi.

Similar content being viewed by others

Change history

  • 09 January 2012

    In the version of this article initially published, information in Table 1 was inaccurate. “Newly described” should have been “novel” and “Argonaute protein domain” should have read “Argonaute protein.” The errors have been corrected in the HTML and PDF versions of the article.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. 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  PubMed  Google Scholar 

  7. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Ketting, R.F. The many faces of RNAi. Dev. Cell 20, 148–161 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Talsky, K.B. & Collins, K. Initiation by a eukaryotic RNA-dependent RNA polymerase requires looping of the template end and is influenced by the template-tailing activity of an associated uridyltransferase. J. Biol. Chem. 285, 27614–27623 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Claycomb, J.M. et al. The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell 139, 123–134 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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  PubMed  Google Scholar 

  14. Gu, W. et al. Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol. Cell 36, 231–244 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rocheleau, C.E. et al. The Caenorhabditis elegans ekl (enhancer of ksr-1 lethality) genes include putative components of a germline small RNA pathway. Genetics 178, 1431–1443 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Conine, C.C. et al. Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 107, 3588–3593 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pavelec, D.M., Lachowiec, J., Duchaine, T.F., Smith, H.E. & Kennedy, S. Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans. Genetics 183, 1283–1295 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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  PubMed  Google Scholar 

  20. Vasale, J.J. et al. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway. Proc. Natl. Acad. Sci. USA 107, 3582–3587 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gent, J.I. et al. Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma. Mol. Cell 37, 679–689 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Welker, N.C. et al. Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs. RNA 16, 893–903 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wolters, D.A., Washburn, M.P. & Yates, J.R. III. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683–5690 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Gu, S.G. et al. Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C. elegans. RNA 13, 1492–1504 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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  PubMed  Google Scholar 

  26. Han, T. et al. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 106, 18674–18679 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Colmenares, S.U., Buker, S.M., Buhler, M., Dlakic, M. & Moazed, D. Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi. Mol. Cell 27, 449–461 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Lee, S.R. & Collins, K. Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs. Nat. Struct. Mol. Biol. 14, 604–610 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Welker, N.C. et al. Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode. Mol. Cell 41, 589–599 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ma, E., MacRae, I.J., Kirsch, J.F. & Doudna, J.A. Autoinhibition of human dicer by its internal helicase domain. J. Mol. Biol. 380, 237–243 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Maurer-Stroh, S. et al. The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28, 69–74 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Thomson, T. & Lasko, P. Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning. Genesis 40, 164–170 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Timmons, L., Court, D.L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Wu, E. et al. Pervasive and cooperative deadenylation of 3′UTRs by embryonic microRNA families. Mol. Cell 40, 558–570 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Raymond, C.K., Roberts, B.S., Garrett-Engele, P., Lim, L.P. & Johnson, J.M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737–1744 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Rocheleau for comments on the manuscript, N. Uetani for conceptual and artistic contributions to the model, S. Mitani and his group at the Department of Physiology, Tokyo Women's Medical University School of Medicine, for the generation of the eri-5(tm2528) allele introduced in this manuscript. We thank I. MacRae and N. Welker for discussions on the ERIC model. We also thank A. Haggarty for her assistance in the development of some of the polyclonal antisera. This work was supported by the National Sciences and Engineering Council of Canada RGPIN 341457 (T.F.D.), the Canadian Institute of Health Research MOP 86577 (T.F.D.), the Canada Foundation for Innovation (C.F.I.), and the Fonds de la Rercherche en Santé du Québec, Chercheur-Boursier Salary Award J2 (T.F.D.).

Author information

Author notes

  1. Caroline Thivierge and Neetha Makil: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, McGill University, Montreal, Quebec, Canada

    Caroline Thivierge, Neetha Makil, Mathieu Flamand & Thomas F Duchaine

  2. Goodman Cancer Center, McGill University, Montreal, Quebec, Canada

    Caroline Thivierge, Neetha Makil, Mathieu Flamand & Thomas F Duchaine

  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jessica J Vasale, Craig C Mello & Darryl Conte Jr

  4. Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Craig C Mello

  5. Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    James Wohlschlegel

Authors
  1. Caroline Thivierge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Neetha Makil
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mathieu Flamand
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jessica J Vasale
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Craig C Mello
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James Wohlschlegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Darryl Conte Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thomas F Duchaine
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.T. conducted the experiments presented in Figures 1c, 2c,d, 3a,b, 4a,b,d and 5a, prepared the figures and assisted with the preparation of the manuscript. N.M. conducted the experiments presented in Figure 1a,d, the ERI-5 samples in 1e and 2a,b. M.F. conducted the experiments presented in Figure 4b,d, and assisted with the model. J.W. carried out the MuDPIT analyses of IP samples. D.C. and J.J.V. conducted the experiments in Figure 3c, under C.C.M.'s direction. D.C. provided scientific advice, and assisted with the redaction of the manuscript. T.F.D. conducted the experiments in Figure 1b, wrote the manuscript and directed the project.

Corresponding author

Correspondence to Thomas F Duchaine.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Results and Supplementary Methods (PDF 4186 kb)

Supplementary Data 1

Supplementary data of the coding loci targeted by 26G-RNAs in eri-5 and rrf-3 embryos. (XLSX 117 kb)

Supplementary Data 2

Complement to Supplementary Figure 3: small RNA defect of eri-5 mutant. (XLSX 1556 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thivierge, C., Makil, N., Flamand, M. et al. Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi. Nat Struct Mol Biol 19, 90–97 (2012). https://doi.org/10.1038/nsmb.2186

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2186

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