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:

The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage

Nature Structural & Molecular Biology volume 12, pages 489–496 (2005)Cite this article

Abstract

Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways. dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I). Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA. These two pathways have previously been shown to be antagonistic. We show a novel interaction between components of the ADAR and RNAi pathways. Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi. Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I·U and U·I pairs. This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.

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: Multiple I·U and U·I pairs are required for efficient cleavage.
Figure 2: Electrophoretic mobility shift assays show that dsRNA substrates that contain multiple I·U or U·I pairs can form an RNA–protein complex.
Figure 3: TSN protein specifically binds to I-dsRNA.
Figure 4: TSN expression coincides with cleavage activity.
Figure 5: Sucrose gradient of X. laevis oocyte extract.
Figure 6: rTSN binds specifically to I-dsRNA.
Figure 7: rTSN enhanced cleavage activity in X. laevis oocyte extract.
Figure 8: pdTp and EGTA inhibit specific cleavage of IIUI dsRNA.

Similar content being viewed by others

References

  1. Maquat, L.E. & Carmichael, G.G. Quality control of mRNA function. Cell 104, 173–176 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Samuel, C.E. Protein-nucleic acid interactions and cellular responses to interferon. Methods 15, 161–165 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Zhou, A., Hassel, B.A. & Silverman, R.H. Expression cloning of 2–5A-dependent RNase: a uniquely regulated mediator of interferon action. Cell 72, 753–765 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Silverman, R.H. 2–5A dependent RNase L: a regulated endoribonuclease in the interferon system. In Ribonucleases: Structure and Functions (eds. D'Alessio, G. & Riordan, J.F.) 515–551 (Academic Press, New York, 1997).

    Chapter  Google Scholar 

  5. Bass, B.L. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Zamore, P.D. Ancient pathways programmed by small RNAs. Science 296, 1265–1269 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Denli, A.M. & Hannon, G.J. RNAi: an ever growing puzzle. Trends Biochem. Sci. 28, 196–201 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Novina, C.D. & Sharp, P.A. The RNAi revolution. Nature 430, 161–164 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Nishikura, K. et al. Substrate specificity of the dsRNA unwinding/modifying activity. EMBO J. 10, 3523–3532 (1991).

  10. Polson, A.G. & Bass, B.L. Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase. EMBO J. 13, 5701–5711 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Serra, M.J., Smolter, P.E. & Westhof, E. Pronounced instability of tandem IU base pairs in RNA. Nucleic Acids Res. 32, 1824–1828 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang, Z. & Carmichael, G.G. The fate of dsRNA in the nucleus: a p54nrb-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell 106, 465–475 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Scadden, A.D.J. & Smith, C.W.J. Specific cleavage of hyper-edited dsRNAs. EMBO J. 20, 4243–4252 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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  PubMed  Google Scholar 

  15. 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).

    Article  CAS  PubMed  Google Scholar 

  16. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Meister, G. et al. Human argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. 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. 101, 14385–14389 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Scadden, A.D.J. & Smith, C.W.J. RNAi is antagonized by A to I hyper-editing. EMBO Rep. 2, 1107–1111 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Knight, S.W. & Bass, B.L. The role of RNA editing by ADARs in RNAi. Mol. Cell 10, 809–817 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Tonkin, L.A. & Bass, B.L. Mutations in RNAi rescue aberrant chemotaxis of ADAR mutants. Science 302, 1725 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Bass, B.L. Double-stranded RNA as a template for gene silencing. Cell 101, 235–238 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Yang, W. et al. ADAR1 RNA deaminase limits siRNA efficacy in mammalian cells. J. Biol. Chem. 280, 3946–3953 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Pharm, 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).

    Article  Google Scholar 

  27. Wollerton, M.C. et al. Differential alternative splicing activity of isoforms of polypyrimidine tract binding proteins (PTB). RNA 7, 819–832 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cuatrecasas, P., Fuchs, S. & Anfinsen, C.B. Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus. J. Biol. Chem. 242, 1541–1547 (1967).

    CAS  PubMed  Google Scholar 

  29. Ponting, C.P. Tudor domains in proteins that interact with RNA. Trends Biochem. Sci. 22, 51–52 (1997).

    Article  CAS  PubMed  Google Scholar 

  30. Callebaut, I. & Mornon, J.P. The human EBNA-2 coactivator p100: multidomain organization and relationship to the staphylococcal nuclease fold and to the Tudor protein involved in Drosophila melanogaster development. Biochem. J. 321, 125–132 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yang, J. et al. Identification of p100 as a coactivator for STAT6 that bridges STAT6 with RNA polymerase II. EMBO J. 21, 4950–4958 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Luciano, D.J., Mirsky, H., Vendetti, N.J. & Mass, S. RNA editing of a miRNA precursor. RNA 10, 1174–1177 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Levanon, E.Y. et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat. Biotechnol. 22, 1001–1005 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  35. Minshall, N. & Standart, N. The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer. Nucleic Acids Res. 32, 1325–1334 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Körner, C.G. et al. The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of X. laevis oocytes. EMBO J. 17, 5427–5437 (1998).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

I would like to thank G. Hannon, S. Holmes, N. Minshall, R. Allison, L. Colegrove, A. Devaux and C. Gooding for gifts of reagents, and the Cambridge Centre for Proteomics (in particular K. Lilley) for protein analyses. I would like to thank C. Smith for helpful discussions and critically reading the manuscript, and also N. Proudfoot and M. O'Connell for comments on the manuscript. This work was supported by a Research Career Development Fellowship from the Wellcome Trust. The Cambridge Centre for Proteomics facility at Cambridge was funded by the UK Biotechnology and Biological Sciences Research Council's Investigating Gene Function Initiative.

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

    A D J Scadden

Authors
  1. A D J Scadden
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A D J Scadden.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Scadden, A. The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage. Nat Struct Mol Biol 12, 489–496 (2005). https://doi.org/10.1038/nsmb936

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb936

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