Abstract
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2–Pan3 and Ccr4–Caf1 deadenylase complexes followed by Dcp1–Dcp2 complex–directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).
Bushati, N. & Cohen, S.M. microRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).
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).
Jackson, R.J. & Standart, N. How do microRNAs regulate gene expression? Sci. STKE 2007, re1 (2007).
Nissan, T. & Parker, R. Computational analysis of miRNA-mediated repression of translation: Implications for models of translation initiation inhibition. RNA 14, 1480–1491 (2008).
Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).
Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).
Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).
Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).
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).
Giraldez, A.J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).
Wu, L., Fan, J. & Belasco, J.G. MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. USA 103, 4034–4039 (2006).
Shyu, A.B. & Chen, A.C.-Y. Regulation of mRNA turnover. in Handbook of Cell Signaling 2nd edn. (eds. Bradshaw, R.A. & Dennis, E.A.) Ch. 277, 2311–2315 (Elsevier, San Diego, 2009).
Hutvágner, G. & Zamore, P.D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002).
Yekta, S., Shih, I.H. & Bartel, D.P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004).
Barth, S. et al. Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. Nucleic Acids Res. 36, 666–675 (2008).
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).
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).
Eulalio, A., Huntzinger, E. & Izaurralde, E. Getting to the root of miRNA-mediated gene silencing. Cell 132, 9–14 (2008).
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).
Ding, L. & Han, M. GW182 family proteins are crucial for microRNA-mediated gene silencing. Trends Cell Biol. 17, 411–416 (2007).
Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol. 8, 1267–1274 (2005).
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. Identificiation of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).
Chu, C.-Y. & Rana, T.M. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 4, e210 (2006).
Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008).
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).
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).
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).
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).
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).
Kiriakidou, M. et al. An mRNA m7G cap binding-like motif within human Ago2 represses translation. Cell 129, 1141–1151 (2007).
Chen, A.C.-Y., Ezzeddine, N. & Shyu, A.B. Messenger RNA half-life measuremments in mammalian cells. Methods Enzymol. 448, 335–357 (2008).
Yamashita, A. et al. Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover. Nat. Struct. Mol. Biol. 12, 1054–1063 (2005).
Zheng, D. et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J. Cell Biol. 182, 89–101 (2008).
Pillai, R.S. et al. Inhibition of translational initiation by let-7 micro-RNA in human cells. Science 309, 1573–1576 (2005).
Baron-Benhamou, J., Gehring, N.H., Kulozik, A.E. & Hentze, M.W. Using the lambda N peptide to tether proteins to RNAs. Methods Mol. Biol. 257, 135–154 (2004).
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).
Liu, J. et al. Argonaute2 Is the Catalytic Engine of Mammalian RNAi. Science 305, 1437–1441 (2004).
Xu, N., Loflin, P., Chen, C.-Y.A. & Shyu, A.-B. A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. Nucleic Acids Res. 26, 558–565 (1998).
Chen, C.-Y.A. & Shyu, A.-B. Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway. Mol. Cell. Biol. 23, 4805–4813 (2003).
Chen, C.-Y.A. & Shyu, A.-B. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol. 14, 8471–8482 (1994).
Chen, C.-Y.A. & Shyu, A.-B. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem. Sci. 20, 465–470 (1995).
Ross, J. mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450 (1995).
Grosset, C. et al. A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 103, 29–40 (2000).
Ponting, C.P., Oliver, P.L. & Reik, W. Evolution and functions of long noncoding RNAs. Cell 136, 629–641 (2009).
Shyu, A.B., Wilkinson, M.F. & van Hoof, A. Messenger RNA regulation: to translate or to degrad. EMBO J. 27, 471–481 (2008).
Eulalio, A. et al. Deadenylation is a widespread effect of miRNA regulation. RNA 15, 21–32 (2009).
Baillat, D. & Shiekhattar, R. Functional dissection of the human TNRC6 (GW182-related) family of proteins. Mol. Cell. Biol. 29, 4144–4155 (2009).
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).
Fabian, M.R. et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol. Cell 35, 868–880 (2009).
von Roretz, C. & Gallouzi, I.-E. Decoding ARE-mediated decay: is microRNA part of the equation? J. Cell Biol. 181, 189–194 (2008).
Wang, Z., Jiao, X., Carr-Schmid, A. & Kiledjian, M. The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc. Natl. Acad. Sci. USA 99, 12663–12668 (2002).
Shyu, A.-B., Belasco, J.G. & Greenberg, M.G. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 5, 221–231 (1991).
Peng, S.-S., Chen, C.-Y.A., Xu, N. & Shyu, A.-B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 17, 3461–3470 (1998).
Acknowledgements
We thank J. Lever for critical reading of the manuscript; W. Filipowicz (Friedrich Miescher Institute, Basel), N. Gehring (EMBL, Heidelberg), G. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA), M. Hentze (European Molecular Biology Laboratory, Heidelberg), M. Kiledjian (Rutgers University, Newark, New Jersey, USA), M. Kiriakidou (University of Pennsylvania, Philadelphia), A. Kulozik (University of Heidelberg), R. Lloyd (Baylor College of Medicine, Houston, Texas, USA) and T. Tuschl (Rockefeller University, New York) for plasmid and antibody supply; and M. Fabian, A. Yamashita, Y. Yamashita, and Y. Zhai for technical assistance. This work was supported by the National Institutes of Health (RO1 GM046454 to A.-B.S.) and in part by the Houston Endowment, Inc. to A.-B.S.
Author information
Authors and Affiliations
Contributions
C.-Y.A.C. designed experiments, analyzed data, and wrote the manuscript; D.Z. and Z.X. performed the experiments; A.-B.S. supervised the project and participated in the manuscript preparation.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 (PDF 795 kb)
Rights and permissions
About this article
Cite this article
Chen, CY., Zheng, D., Xia, Z. et al. Ago–TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps. Nat Struct Mol Biol 16, 1160–1166 (2009). https://doi.org/10.1038/nsmb.1709
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1709
This article is cited by
-
Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events
Genome Biology (2023)
-
A genome-wide association study of tinnitus reveals shared genetic links to neuropsychiatric disorders
Scientific Reports (2022)
-
MicroRNAs involve in bicuspid aortic aneurysm: pathogenesis and biomarkers
Journal of Cardiothoracic Surgery (2021)
-
Aedes aegypti miRNA-33 modulates permethrin induced toxicity by regulating VGSC transcripts
Scientific Reports (2021)
-
eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
Genome Biology (2019)