Abstract
miRNAs recruit the miRNA-induced silencing complex (miRISC), which includes Argonaute and GW182 as core proteins. GW182 proteins effect translational repression and deadenylation of target mRNAs. However, the molecular mechanisms of GW182-mediated repression remain obscure. We show here that human GW182 independently interacts with the PAN2–PAN3 and CCR4–NOT deadenylase complexes. Interaction of GW182 with CCR4–NOT is mediated by two newly discovered phylogenetically conserved motifs. Although either motif is sufficient to bind CCR4–NOT, only one of them can promote processive deadenylation of target mRNAs. Thus, GW182 serves as both a platform that recruits deadenylases and as a deadenylase coactivator that facilitates the removal of the poly(A) tail by CCR4–NOT.
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Change history
08 November 2011
In the version of this article initially published, several acknowledgments were missing. The end of the section should read as follows: "...N.S., who was also supported by a Howard Hughes Medical Institute (HHMI) International Scholarship. Postdoctoral support was from the Terry Fox Foundation of the Canadian Cancer Society to M.R.F. T.F.D. was supported through a CIHR grant. M.K.C. was supported by the Foundation for Polish Science International PhD Projects. F.F. was supported by a Boehringer Ingelheim Fonds PhD fellowship." The error has been corrected in the HTML and PDF versions of the article.
References
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).
Huntzinger, E. & Izaurralde, E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat. Rev. Genet. 12, 99–110 (2011).
Fabian, M.R., Sonenberg, N. & Filipowicz, W. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem. 79, 351–379 (2010).
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).
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).
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).
Jakymiw, A. et al. The role of GW/P-bodies in RNA processing and silencing. J. Cell Sci. 120, 1317–1323 (2007).
Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nat. Cell Biol. 7, 1261–1266 (2005).
Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).
Zipprich, J.T., Bhattacharyya, S., Mathys, H. & Filipowicz, W. Importance of the C-terminal domain of the human GW182 protein TNRC6C for translational repression. RNA 15, 781–793 (2009).
Chekulaeva, M., Parker, R. & Filipowicz, W. The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression. Nucleic Acids Res. 38, 6673–6683 (2010).
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).
Lazzaretti, D., Tournier, I. & Izaurralde, E. The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins. RNA 15, 1059–1066 (2009).
Baillat, D. & Shiekhattar, R. Functional dissection of the human TNRC6 (GW182-related) family of proteins. Mol. Cell Biol. 29, 4144–4155 (2009).
Jinek, M., Fabian, M.R., Coyle, S.M., Sonenberg, N. & Doudna, J.A. Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation. Nat. Struct. Mol. Biol. 17, 238–240 (2010).
Eulalio, A., Tritschler, F. & Izaurralde, E. The GW182 protein family in animal cells: new insights into domains required for miRNA-mediated gene silencing. RNA 15, 1433–1442 (2009).
Takimoto, K., Wakiyama, M. & Yokoyama, S. Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression. RNA 15, 1078–1089 (2009).
Lian, S.L. et al. The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing. RNA 15, 804–813 (2009).
Eulalio, A. et al. The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing. Nucleic Acids Res. 37, 2974–2983 (2009).
Huntzinger, E., Braun, J.E., Heimstadt, S., Zekri, L. & Izaurralde, E. Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing. EMBO J. 29, 4146–4160 (2010).
Zekri, L., Huntzinger, E., Heimstadt, S. & Izaurralde, E. The silencing domain of GW182 interacts with PABPC1 to promote translational repression and degradation of microRNA targets and is required for target release. Mol. Cell Biol. 29, 6220–6231 (2009).
Kozlov, G., Safaee, N., Rosenauer, A. & Gehring, K. Structural basis of binding of P-body-associated proteins GW182 and ataxin-2 by the Mlle domain of poly(A)-binding protein. J. Biol. Chem. 285, 13599–13606 (2010).
Fabian, M.R. et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol. Cell 35, 868–880 (2009).
Goldstrohm, A.C. & Wickens, M. Multifunctional deadenylase complexes diversify mRNA control. Nat. Rev. Mol. Cell Biol. 9, 337–344 (2008).
Mathonnet, G. et al. MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science 317, 1764–1767 (2007).
Wu, E. et al. Pervasive and cooperative deadenylation of 3′UTRs by embryonic microRNA families. Mol. Cell 40, 558–570 (2010).
Siddiqui, N. et al. Poly(A) nuclease interacts with the C-terminal domain of polyadenylate-binding protein domain from poly(A)-binding protein. J. Biol. Chem. 282, 25067–25075 (2007).
Uchida, N., Hoshino, S. & Katada, T. Identification of a human cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein. J. Biol. Chem. 279, 1383–1391 (2004).
Brown, C.E., Tarun, S.Z. Jr., Boeck, R. & Sachs, A.B. PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae. Mol. Cell Biol. 16, 5744–5753 (1996).
Lau, N.C. et al. Human Ccr4-Not complexes contain variable deadenylase subunits. Biochem. J. 422, 443–453 (2009).
Piao, X., Zhang, X., Wu, L. & Belasco, J.G. CCR4-NOT deadenylates mRNA associated with RNA-induced silencing complexes in human cells. Mol. Cell Biol. 30, 1486–1494 (2010).
Chen, C.Y., Zheng, D., Xia, Z. & Shyu, A.B. Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps. Nat. Struct. Mol. Biol. 16, 1160–1166 (2009).
Zheng, D. et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J. Cell Biol. 182, 89–101 (2008).
Simón, E. & Seraphin, B. A specific role for the C-terminal region of the Poly(A)-binding protein in mRNA decay. Nucleic Acids Res. 35, 6017–6028 (2007).
Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D. & Parker, R. Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J. 21, 1427–1436 (2002).
Baer, B.W. & Kornberg, R.D. Repeating structure of cytoplasmic poly(A)-ribonucleoprotein. Nat. Struct. Mol. Biol 77, 1890–1892 (1980).
Chekulaeva, M. et al. miRNA repression involves GW182‐mediated recruitment of CCR4–NOT through conserved W-containing motifs. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.2166 (published online 7 Oct 2011).
Braun, J.E., Huntzinger, E., Fauser, M. & Izaurralde, E. GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets. Mol. Cell 43, doi:10.1016/j.molcel.2011.09.007 (2011).
Jinek, M., Fabian, M.R., Coyle, S.M., Sonenberg, N. & Doudna, J.A. Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation. Nat. Struct. Mol. Biol. 17, 238–240 (2010).
Ito, K. et al. CNOT2 depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death. Genes Cells 16, 368–379 (2011).
Miyasaka, T. et al. Interaction of antiproliferative protein Tob with the CCR4-NOT deadenylase complex. Cancer Sci. 99, 755–761 (2008).
Morita, M. et al. Depletion of mammalian CCR4b deadenylase triggers elevation of the p27Kip1 mRNA level and impairs cell growth. Mol. Cell Biol. 27, 4980–4990 (2007).
Craig, R., Cortens, J.P. & Beavis, R.C. Open source system for analyzing, validating, and storing protein identification data. J. Proteome Res. 3, 1234–1242 (2004).
Fabian, M.R. et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol. Cell 35, 868–880 (2009).
Acknowledgements
We thank G. Hannon (Cold Spring Harbor Laboratory) for providing rabbit polyclonal CNOT10 antibody; A.-B. Shyu (University of Texas Medical School) for rabbit polyclonal PAN2 antibody; W. Filipowicz (Friedrich Miescher Institute for Biomedical Research) for TNRC6C and LacZ plasmids; and M. Jinek, T. Sundermeier and I. Topisirovic for helpful discussions. This work was supported by a Canadian Institutes of Health Research (CIHR) grant to N.S., who was also supported by a Howard Hughes Medical Institute (HHMI) International Scholarship. Postdoctoral support was from the Terry Fox Foundation of the Canadian Cancer Society to M.R.F. T.F.D. was supported through a CIHR grant. M.K.C. was supported by the Foundation for Polish Science International PhD Projects. F.F. was supported by a Boehringer Ingelheim Fonds PhD fellowship.
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M.R.F., T.F.D. and N.S. designed experiments. M.R.F., M.K.C., J.G. and M.M. conducted experiments. T.S. and B.R. conducted mass spectrometry analysis. F.F. and B.N. purified recombinant CNOT1 protein. T.Y. provided CNOT antibodies. M.R.F., T.F.D. and N.S. wrote the manuscript.
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Fabian, M., Cieplak, M., Frank, F. et al. miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4–NOT. Nat Struct Mol Biol 18, 1211–1217 (2011). https://doi.org/10.1038/nsmb.2149
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DOI: https://doi.org/10.1038/nsmb.2149
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