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:

Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins

Nature Structural & Molecular Biology volume 16, pages 1259–1266 (2009)Cite this article

Abstract

Argonaute (AGO) proteins bind to small RNAs and mediate small RNA−induced silencing in eukaryotes. Using a minimal in vitro system, we show that bacterially expressed human AGO1 and AGO2 but not AGO3 and AGO4 possess strand-dissociating activity of microRNA (miRNA) duplexes. Both AGO1 and AGO2 function as RNA chaperones, capable of performing multiple rounds of strand dissociation. Unexpectedly, both AGO1 and AGO2 demonstrate passenger strand cleavage activity of a small interfering RNA (siRNA) duplex, but only AGO2 has target RNA cleavage activity. These observations indicate that passenger strand and mRNA endonuclease activities are mechanistically distinct. We further validate these observations in mammalian extracts and cultured mammalian cells, in which we demonstrate that AGO1 uses only miRNA duplexes when assembling translational repression−competent complexes, whereas AGO2 can use both miRNA and siRNA duplexes. We show that passenger strand cleavage and RNA chaperone activities that are intrinsic to both AGO1 and AGO2 are sufficient for RNA-induced silencing complex (RISC) loading.

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: AGO1 and AGO2 possess passenger strand cleavage activity toward miR-21P duplexes.
Figure 2: AGO1 and AGO2 possess strand-dissociating activity toward miR-21−miR-21* duplexes.
Figure 3: AGO chimeras possess passenger strand cleavage activity of siRNA duplexes but lack strand-dissociating activity of miRNA duplexes.
Figure 4: miRNA−miRNA* but not siRNA duplexes load into mRNA cleavage and translational repression−competent complexes in vitro.
Figure 5: AGO1 and AGO2 load miR-21−miR-21* duplexes into functional RISC in cells.
Figure 6: RNA chaperone model of AGO1- and AGO2-mediated RISC loading.

Similar content being viewed by others

References

  1. Filipowicz, W., Bhattacharyya, S.N. & Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102–114 (2008).

    Article  CAS  Google Scholar 

  2. 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 

  3. 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 

  4. 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 

  5. 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).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. 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 

  14. Robb, G.B. & Rana, T.M. RNA helicase A interacts with RISC in human cells and functions in RISC loading. Mol. Cell 26, 523–537 (2007).

    Article  CAS  Google Scholar 

  15. Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).

    Article  CAS  Google Scholar 

  16. Chu, C.Y. & Rana, T.M. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 4, e210 (2006).

    Article  Google Scholar 

  17. Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728 (2002).

    Article  CAS  Google Scholar 

  18. 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 

  19. 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 

  20. Gregory, R.I., Chendrimada, T.P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Miyoshi, K., Uejima, H., Nagami-Okada, T., Siomi, H. & Siomi, M.C. In vitro RNA cleavage assay for Argonaute-family proteins. Methods Mol. Biol. 442, 29–43 (2008).

    Article  CAS  Google Scholar 

  23. Doench, J.G., Petersen, C.P. & Sharp, P.A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).

    Article  CAS  Google Scholar 

  24. Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    Article  CAS  Google Scholar 

  25. 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 

  26. Haley, B. & Zamore, P.D. Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606 (2004).

    Article  CAS  Google Scholar 

  27. Wang, B., Love, T.M., Call, M.E., Doench, J.G. & Novina, C.D. Recapitulation of short RNA-directed translational gene silencing in vitro. Mol. Cell 22, 553–560 (2006).

    Article  CAS  Google Scholar 

  28. Wang, B., Yanez, A. & Novina, C.D. MicroRNA-repressed mRNAs contain 40S, but not 60S components. Proc. Natl. Acad. Sci. USA 105, 5343–5348 (2008).

    Article  CAS  Google Scholar 

  29. Griffiths-Jones, S. The MicroRNA Registry. Nucleic Acids Res. 32, D109–D111 (2004).

    Article  CAS  Google Scholar 

  30. Zeng, Y. Regulation of the mammalian nervous system by microRNAs. Mol. Pharmacol. 75, 259–264 (2009).

    Article  CAS  Google Scholar 

  31. Qi, H.H. et al. Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 455, 421–424 (2008).

    Article  CAS  Google Scholar 

  32. Beitzinger, M., Peters, L., Zhu, J.Y., Kremmer, E. & Meister, G. Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol. 4, 76–84 (2007).

    Article  CAS  Google Scholar 

  33. Easow, G., Teleman, A.A. & Cohen, S.M. Isolation of microRNA targets by miRNP immunopurification. RNA 13, 1198–1204 (2007).

    Article  CAS  Google Scholar 

  34. Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008).

    Article  CAS  Google Scholar 

  35. Hendrickson, D.G., Hogan, D.J., Herschlag, D., Ferrell, J.E. & Brown, P.O. Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance. PLoS One 3, e2126 (2008).

    Article  Google Scholar 

  36. Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  Google Scholar 

  37. Asangani, I.A. et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27, 2128–2136 (2008).

    Article  CAS  Google Scholar 

  38. Frankel, L.B. et al. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J. Biol. Chem. 283, 1026–1033 (2008).

    Article  CAS  Google Scholar 

  39. Lu, Z. et al. MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene. Oncogene 27, 4373–4379 (2008).

    Article  CAS  Google Scholar 

  40. Zhu, S. et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 18, 350–359 (2008).

    Article  CAS  Google Scholar 

  41. Nelson, P.T., Hatzigeorgiou, A.G. & Mourelatos, Z. miRNP:mRNA association in polyribosomes in a human neuronal cell line. RNA 10, 387–394 (2004).

    Article  CAS  Google Scholar 

  42. Höck, J. et al. Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells. EMBO Rep. 8, 1052–1060 (2007).

    Article  Google Scholar 

  43. 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 

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

    Article  CAS  Google Scholar 

  45. Rajkowitsch, L. et al. RNA chaperones, RNA annealers and RNA helicases. RNA Biol. 4, 118–130 (2007).

    Article  CAS  Google Scholar 

  46. Izzo, A., Regnard, C., Morales, V., Kremmer, E. & Becker, P.B. Structure-function analysis of the RNA helicase maleless. Nucleic Acids Res. 36, 950–962 (2008).

    Article  CAS  Google Scholar 

  47. Wang, B., Doench, J.G. & Novina, C.D. Analysis of microRNA effector functions in vitro. Methods 43, 91–104 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We thank M. Janas and J. Doench for insightful recommendations and critical reading of the manuscript. We also thank Y. Pan for assistance with the AGO pull-down assays. We thank E. Gagnon for creating Figure 6. This work was supported by a Distinguished Young Scholars Award from the W.M. Keck Foundation to C.D.N.

Author information

Authors and Affiliations

  1. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Bingbing Wang, Shuqiang Li & Carl D Novina

  2. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Bingbing Wang, Shuqiang Li, Hank H Qi, Yang Shi & Carl D Novina

  3. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Dipanjan Chowdhury

  4. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Dipanjan Chowdhury

  5. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    Carl D Novina

Authors
  1. Bingbing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuqiang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hank H Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dipanjan Chowdhury
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yang Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carl D Novina
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.W. designed and performed experiments, analyzed data and co-wrote the manuscript; S.L. and H.H.Q. performed experiments; D.C. and Y.S. analyzed data; C.D.N. designed experiments, analyzed data and co-wrote the manuscript.

Corresponding author

Correspondence to Carl D Novina.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Discussion (PDF 9647 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, B., Li, S., Qi, H. et al. Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins. Nat Struct Mol Biol 16, 1259–1266 (2009). https://doi.org/10.1038/nsmb.1712

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1712

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