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GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay

Nature Structural & Molecular Biology volume 15, pages 346–353 (2008)Cite this article

Abstract

MicroRNAs (miRNAs) silence gene expression by binding 3′ untranslated regions of target mRNAs. Recent studies suggested silencing is achieved through either recruitment of eIF6, which prevents ribosome assembly, or displacement of eIF4E from the mRNA 5′ cap structure. Using Drosophila melanogaster cells, we show that eIF6 is not required for silencing. In contrast, silencing is abolished by mutating Argonaute 1 (AGO1) at two conserved phenylalanine residues predicted to mediate binding to the cap structure. Notably, we found these mutations also prevented AGO1 from interacting with GW182 and miRNAs, indicating that the essential role of these residues is unrelated to cap binding. Consistently, depleting GW182 or overexpressing its AGO1 binding domain relieved silencing of all reporters tested, including those lacking a poly(A) tail. Together, our findings show that miRNA function is effected by AGO1–GW182 complexes and the role of GW182 in silencing goes beyond promoting deadenylation.

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Figure 1: eIF6 is not generally required for miRNA-mediated gene silencing.
Figure 2: eIF6 is not generally required for miRNA-mediated gene silencing.
Figure 3: Valine substitutions of Phe594 and Phe629 in the mid domain of AGO1 abrogate silencing by miRNAs.
Figure 4: Conserved phenylalanine residues in the mid domain of Argonaute proteins are required for silencing in tethering assays.
Figure 5: Valine substitutions of Phe594 and Phe629 abolish the AGO1 interaction with miRNAs and GW182.
Figure 6: Overexpression of the AGO1 binding domain of GW182 supresses miRNA-mediated silencing.
Figure 7: GW182 is required for silencing of nonadenylated mRNAs.

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References

  1. Bushati, N. & Cohen, S.M. MicroRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).

    Article  CAS  Google Scholar 

  2. Nilsen, T.W. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet. 23, 243–249 (2007).

    Article  CAS  Google Scholar 

  3. Pillai, R.S., Bhattacharyya, S.N. & Filipowicz, W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 17, 118–126 (2007).

    Article  CAS  Google Scholar 

  4. Eulalio, A., Huntzinger, E. & Izaurralde, E. Getting to the root of miRNA-mediated gene silencing. Cell 132, 9–14 (2008).

    Article  CAS  Google Scholar 

  5. Chendrimada, T.P. et al. MicroRNA silencing through RISC recruitment of eIF6. Nature 447, 823–828 (2007).

    Article  CAS  Google Scholar 

  6. Ceci, M. et al. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426, 579–584 (2003).

    Article  CAS  Google Scholar 

  7. Basu, U., Si, K., Warner, J.R. & Maitra, U. The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis. Mol. Cell. Biol. 21, 1453–1462 (2001).

    Article  CAS  Google Scholar 

  8. Volta, V. et al. Sen34p depletion blocks tRNA splicing in vivo and delays rRNA processing. Biochem. Biophys. Res. Commun. 337, 89–94 (2005).

    Article  CAS  Google Scholar 

  9. Kiriakidou, M. et al. An mRNA m(7)G Cap binding-like motif within human Ago2 represses translation. Cell 129, 1141–1151 (2007).

    Article  CAS  Google Scholar 

  10. 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 

  11. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).

    Article  CAS  Google Scholar 

  12. Wu, L. & Belasco, J.G. Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. Mol. Cell. Biol. 25, 9198–9208 (2005).

    Article  CAS  Google Scholar 

  13. Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885–1898 (2006).

    Article  CAS  Google Scholar 

  14. Behm-Ansmant, I., Rehwinkel, J. & Izaurralde, E. MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb. Symp. Quant. Biol. 71, 523–530 (2006).

    Article  CAS  Google Scholar 

  15. Giraldez, A.J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).

    Article  CAS  Google Scholar 

  16. Mishima, Y. et al. Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr. Biol. 16, 2135–2142 (2006).

    Article  CAS  Google Scholar 

  17. 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 

  18. Wu, L., Fan, J. & Belasco, J.G. MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. USA 103, 4034–4039 (2006).

    Article  CAS  Google Scholar 

  19. Eulalio, A. et al. Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing. Genes Dev. 21, 2558–2570 (2007).

    Article  CAS  Google Scholar 

  20. 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 

  21. 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 

  22. Grivna, S.T., Pyhtila, B. & Lin, H. MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis. Proc. Natl. Acad. Sci. USA 103, 13415–13420 (2006).

    Article  CAS  Google Scholar 

  23. 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 

  24. Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol. 7, 1167–1174 (2005).

    Article  CAS  Google Scholar 

  25. 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 

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

    Article  CAS  Google Scholar 

  27. 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 

  28. Zhang, L. et al. Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol. Cell 28, 598–613 (2007).

    Article  CAS  Google Scholar 

  29. 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 

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

    Article  CAS  Google Scholar 

  31. El-Shami, M. et al. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev. 21, 2539–2544 (2007).

    Article  CAS  Google Scholar 

  32. 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 

  33. 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 

  34. 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 

  35. 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 

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Acknowledgements

We are grateful to S.M. Cohen (Temasek Life Sciences Laboratory, Singapore) for providing miRNA expression vectors and the nerfin-1 and par-6 reporters, S. Dorner (University of Vienna) for generating reporters containing the histone H4 stem-loop structure, W. Filipowicz (Friedich Miescher Institute, Basel, Switzerland) for plasmids for tethering assays in human cells, and J. Pelletier (McGill University, Montreal) for his generous gift of hippuristanol. This study was supported by the Max Planck Society, by a grant from the Deutsche Forschungsgemeinschaft (DFG, FOR855) and by the Sixth Framework Programme of the European Commission through the SIROCCO Integrated Project LSHG-CT-2006-037900. A.E. and E.H. are recipients of fellowships from the Portuguese Foundation for Science and Technology and the European Molecular Biology Organization (EMBO), respectively.

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  1. Max Planck Institute for Developmental Biology, Spemannstrasse 35, Tübingen, D-72076, Germany

    Ana Eulalio, Eric Huntzinger & Elisa Izaurralde

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  1. Ana Eulalio
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A.E. and E.H. performed all the experiments and analyzed the data. E.I. supervised the research and wrote the manuscript.

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Correspondence to Elisa Izaurralde.

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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). https://doi.org/10.1038/nsmb.1405

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