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miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Nature Structural & Molecular Biology volume 18, pages 1218–1226 (2011)Cite this article

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Abstract

miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4–NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4–NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4–NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation.

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Figure 1: The TNRC6C CED interacts with components of the CCR4–NOT complex.
Figure 2: W-motifs in GW182 proteins mediate mRNA repression by recruiting CCR4–NOT and PAN2–PAN3 deadenylation complexes.
Figure 3: W-motifs present in the dGW182 NED and the engineered yeast protein fragment repress tethered mRNA and recruit components of CCR4–NOT.
Figure 4: W-motifs are necessary for repression by full-length GW182 and function in bona fide miRNA repression.
Figure 5: The CED W-motifs and CCR4–NOT complex contribute to repression of poly(A)− mRNAs in fly cells.
Figure 6: Repression of poly(A)− RNA by tethering dGW182 or its fragments depends on NOT1, but repression by tethered CCR4–NOT components is dGW182-independent.

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Acknowledgements

We thank M. Collart (University of Geneva), A.-B. Shyu (University of Texas Medical School), H.T.M. Timmers (University of Utrecht), E. Izaurralde (Max Planck Institute for Developmental Biology), N. Sonenberg (McGill University), K. Schönig (Zentralinstitut für Seelische Gesundheit), J. Bethune (Friedrich Miescher Institute (FMI)), E. Wahle (Martin Luther University, Halle) and T. Yamamoto (University of Tokyo) for reagents; R. Sack, D. Klei, and the FMI Protein Analysis Facility for MS analysis; M. Tsai for help with the Y2H assay; and H. Gut, I. Loedige, J. Krol, J. Bethune, M. de la Mata, N. Thoma, F. Allain, E. Izaurralde and N. Sonenberg for stimulating discussions. M. Chekulaeva is the recipient of long-term postdoctoral fellowships from the Human Frontiers Science Program and Engelhorn Stiftung. J.A. is funded by the German National Academic Foundation. R.P. is supported by funds from the Howard Hughes Medical Institute. This work was supported by the European Community FP6 Program 'Sirocco'. The FMI is supported by the Novartis Research Foundation.

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Author notes

  1. Marina Chekulaeva and Hansruedi Mathys: These authors contributed equally to this work.

Authors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Marina Chekulaeva, Hansruedi Mathys, Jakob T Zipprich, Jan Attig, Marija Colic & Witold Filipowicz

  2. University of Basel, Basel, Switzerland

    Hansruedi Mathys, Jakob T Zipprich & Witold Filipowicz

  3. Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona, USA

    Roy Parker

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  1. Marina Chekulaeva
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Contributions

M. Chekulaeva, H.M., J.T.Z., J.A., M. Colic, R.P. and W.F. designed the experiments. M. Chekulaeva, H.M., J.T.Z., J.A. and M. Colic conducted the experiments. M. Chekulaeva, H.M., R.P. and W.F. wrote the manuscript.

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Correspondence to Witold Filipowicz.

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Chekulaeva, M., Mathys, H., Zipprich, J. et al. miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs. Nat Struct Mol Biol 18, 1218–1226 (2011). https://doi.org/10.1038/nsmb.2166

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