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Ago–TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps

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.

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Figure 1: Let-7 miRMD is triggered by deadenylation followed by decapping.
Figure 2: Decay kinetics of mRNA carrying a sequence that perfectly matches let-7 miRNA.
Figure 3: Tethering Ago2 to mRNAs triggers biphasic deadenylation and decay.
Figure 4: Tethering Ago2 or TNRC6C to mRNAs is sufficient to recapitulate miRMD.
Figure 5: A model for miRMD in mammalian cells.

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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.

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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.

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Correspondence to Ann-Bin Shyu.

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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

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