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:

Allosteric regulation of Argonaute proteins by miRNAs

Nature Structural & Molecular Biology volume 17, pages 144–150 (2010)Cite this article

Subjects

Abstract

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) bind to Argonaute (AGO) family proteins to form a related set of effector complexes that have diverse roles in post-transcriptional gene regulation throughout the eukaryotic lineage. Here sequence and structural analysis of the MID domain of the AGO proteins identified similarities with a family of allosterically regulated bacterial ligand-binding domains. We used in vitro and in vivo approaches to show that certain AGO proteins (those involved in translational repression) have conserved this functional allostery between two distinct sites, one involved in binding miRNA–target duplex and the other in binding the 5′ cap feature (m7GpppG) of eukaryotic mRNAs. This allostery provides an explanation for how miRNA-bound effector complexes may avoid indiscriminate repressive action (mediated through binding interactions with the cap) before full target recognition.

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: Bioinformatic analysis of AGO proteins.
Figure 2: Binding of AGO proteins to m7GTP-Sepharose reveals allosteric behavior.
Figure 3: Filter binding assays indicate two allosterically regulated nucleotide binding sites.
Figure 4: Mutational analysis of AGO proteins in Drosophila S2 cells.
Figure 5: Cartoon describing potential role of AGO allostery in promoting translational repression.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Ghildiyal, M. & Zamore, P.D. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009).

    Article  CAS  Google Scholar 

  2. Carthew, R.W. & Sontheimer, E.J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009).

    Article  CAS  Google Scholar 

  3. Wu, L. & Belasco, J.G. Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs. Mol. Cell 29, 1–7 (2008).

    Article  Google Scholar 

  4. Kiriakidou, M. et al. An mRNA m7G cap binding–like motif within human AGO2 represses translation. Cell 129, 1141–1151 (2007).

    Article  CAS  Google Scholar 

  5. 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 

  6. Humphreys, D.T., Westman, B.J., Martin, D.I. & Preiss, T. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc. Natl. Acad. Sci. USA 102, 16961–16966 (2005).

    Article  CAS  Google Scholar 

  7. Wakiyama, M., Takimoto, K., Ohara, O. & Yokoyama, S. Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system. Genes Dev. 21, 1857–1862 (2007).

    Article  CAS  Google Scholar 

  8. Zdanowicz, A. et al. Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression. Mol. Cell 35, 881–888 (2009).

    Article  CAS  Google Scholar 

  9. 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 

  10. 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 

  11. Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. & Izaurralde, E. A crucial role for GW182 and the DCP1: DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11, 1640–1647 (2005).

    Article  CAS  Google Scholar 

  12. Eulalio, A., Helms, S., Fritzsch, C., Fauser, M. & Izaurralde, E. A C-terminal silencing domain in GW182 is essential for miRNA function. RNA 15, 1067–1077 (2009).

    Article  CAS  Google Scholar 

  13. Chekulaeva, M., Filipowicz, W. & Parker, R. Multiple independent domains of dGW182 function in miRNA-mediated repression in Drosophila. RNA 15, 794–803 (2009).

    Article  CAS  Google Scholar 

  14. Soding, J., Biegert, A. & Lupas, A.N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  Google Scholar 

  15. 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 

  16. Marcotrigiano, J., Gingras, A.C., Sonenberg, N. & Burley, S.K. Cocrystal structure of the messenger RNA 5′ cap–binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961 (1997).

    Article  CAS  Google Scholar 

  17. Holm, L. & Sander, C. Protein-structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  18. Anantharaman, V. & Aravind, L. Diversification of catalytic activities and ligand interactions in the protein fold shared by the sugar isomerases, elF2B, DeoR transcription factors Acyl-CoA transferases and methenyltetrahydrofolate synthetase. J. Mol. Biol. 356, 823–842 (2006).

    Article  CAS  Google Scholar 

  19. Frickey, T. & Lupas, A. CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 20, 3702–3704 (2004).

    Article  CAS  Google Scholar 

  20. Carmell, M.A., Xuan, Z., Zhang, M.Q. & Hannon, G.J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742 (2002).

    Article  CAS  Google Scholar 

  21. Parker, J.S. & Barford, D. Argonaute: a scaffold for the function of short regulatory RNAs. Trends Biochem. Sci. 31, 622–630 (2006).

    Article  CAS  Google Scholar 

  22. Mi, S. et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008).

    Article  CAS  Google Scholar 

  23. Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Till, S. et al. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat. Struct. Mol. Biol. 14, 897–903 (2007).

    Article  CAS  Google Scholar 

  26. Ding, L., Spencer, A., Morita, K. & Han, M. The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol. Cell 19, 437–447 (2005).

    Article  CAS  Google Scholar 

  27. Schumacher, M.A., Choi, K.Y., Zalkin, H. & Brennan, R.G. Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by α helices. Science 266, 763–770 (1994).

    Article  CAS  Google Scholar 

  28. Ma, J.B. et al. Structural basis for 5′-end–specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005).

    Article  CAS  Google Scholar 

  29. Parker, J.S., Roe, S.M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005).

    Article  CAS  Google Scholar 

  30. Eulalio, A., Huntzinger, E. & Izaurralde, E. GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat. Struct. Mol. Biol. 15, 346–353 (2008).

    Article  CAS  Google Scholar 

  31. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 (2001).

    Article  CAS  Google Scholar 

  32. Pillai, R.S., Artus, C.G. & Filipowicz, W. Tethering of human AGO proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. Rna 10, 1518–1525 (2004).

    Article  CAS  Google Scholar 

  33. Rehwinkel, J. et al. Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster. Mol. Cell. Biol. 26, 2965–2975 (2006).

    Article  CAS  Google Scholar 

  34. 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 

  35. Kinch, L.N. & Grishin, N.V. The human AGO2 MC region does not contain an eIF4E-like mRNA cap binding motif. Biol. Direct 4, 2 (2009).

    Article  Google Scholar 

  36. Mathonnet, G. et al. MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science 317, 1764–1767 (2007).

    Article  CAS  Google Scholar 

  37. Miyoshi, K., Okada, T.N., Siomi, H. & Siomi, M.C. Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA 15, 1282–1291 (2009).

    Article  CAS  Google Scholar 

  38. 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 

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

    Article  CAS  Google Scholar 

  40. Vasudevan, S., Tong, Y. & Steitz, J.A. Cell-cycle control of microRNA-mediated translation regulation. Cell Cycle 7, 1545–1549 (2008).

    Article  CAS  Google Scholar 

  41. Tu, B.P. et al. Cyclic changes in metabolic state during the life of a yeast cell. Proc. Natl. Acad. Sci. USA 104, 16886–16891 (2007).

    Article  CAS  Google Scholar 

  42. Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nat. Cell Biol. 7, 1261–1266 (2005).

    Article  Google Scholar 

  43. Liu, J., Valencia-Sanchez, M.A., Hannon, G.J. & Parker, R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, 719–723 (2005).

    Article  CAS  Google Scholar 

  44. Pauley, K.M. et al. Formation of GW bodies is a consequence of microRNA genesis. EMBO Rep. 7, 904–910 (2006).

    Article  CAS  Google Scholar 

  45. Turnbough, C.L., Jr. & Switzer, R.L. Regulation of pyrimidine biosynthetic gene expression in bacteria: repression without repressors. Microbiol. Mol. Biol. Rev. 72, 266–300 (2008).

    Article  CAS  Google Scholar 

  46. Wang, Y.L. 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 

  47. Wang, Y.L., 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 

  48. Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in AGO silencing complexes. Nature 461, 754–761 (2009).

    Article  CAS  Google Scholar 

  49. Guang, S. et al. An Argonaute transports siRNAs from the cytoplasm to the nucleus. Science 321, 537–541 (2008).

    Article  CAS  Google Scholar 

  50. Ginalski, K., Elofsson, A., Fischer, D. & Rychlewski, L. 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19, 1015–1018 (2003).

    Article  CAS  Google Scholar 

  51. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  Google Scholar 

  52. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).

    Article  CAS  Google Scholar 

  53. 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 

  54. Nahvi, A., Shoemaker, C.J. & Green, R. An expanded seed sequence definition accounts for full regulation of the hid 3′ UTR by bantam miRNA. RNA 15, 814–822 (2009).

    Article  CAS  Google Scholar 

  55. Cong, P.J. & Shuman, S. Mutational analysis of messenger RNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. Mol. Cell. Biol. 15, 6222–6231 (1995).

    Article  CAS  Google Scholar 

  56. Stockley, P.G. Filter-binding assays. Methods Mol. Biol. 543, 1–14 (2009).

    Article  CAS  Google Scholar 

  57. O'Hara, B.P. et al. Crystal structure and induction mechanism of AmiC–AmiR: a ligand-regulated transcription antitermination complex. EMBO J. 18, 5175–5186 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Dorner for early contributions to the project, E. Izaurralde (Max Planck Institute) for providing the luciferase reporter constructs, H. Zaher (Johns Hopkins Univ. School of Medicine) for mRNA constructs used in filter binding assays, and J. Mendell, G. Seydoux, J. Lorsch, L. Cochella and H. Zaher for helpful comments on the manuscript. J.K.H. was supported by The Samsung Foundation of Culture. The project was supported by funding from the Howard Hughes Medical Institute.

Author information

Author notes

  1. Sergej Djuranovic and Michelle Kim Zinchenko: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Sergej Djuranovic, Michelle Kim Zinchenko, Junho K Hur, Ali Nahvi, Julie L Brunelle, Elizabeth J Rogers & Rachel Green

Authors
  1. Sergej Djuranovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle Kim Zinchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junho K Hur
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ali Nahvi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Julie L Brunelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elizabeth J Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rachel Green
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.D. performed the bioinformatic analyses and in vitro studies; M.K.Z. performed the in vivo studies; J.K.H., A.N., J.L.B. and E.J.R. performed biochemistry in support of the main experiments; R.G. advised on the project; S.D., M.K.Z. and R.G. prepared the manuscript.

Corresponding author

Correspondence to Rachel Green.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Methods (PDF 1899 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Djuranovic, S., Zinchenko, M., Hur, J. et al. Allosteric regulation of Argonaute proteins by miRNAs. Nat Struct Mol Biol 17, 144–150 (2010). https://doi.org/10.1038/nsmb.1736

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1736

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