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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 346353 (2008) | Download Citation

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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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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, D-72076 Tübingen, Germany.

    • Ana Eulalio
    • , Eric Huntzinger
    •  & Elisa Izaurralde

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Contributions

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

Corresponding author

Correspondence to Elisa Izaurralde.

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DOI

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