Abstract
Polyadenylated mRNAs are typically more strongly repressed by microRNAs (miRNAs) than their nonadenylated counterparts. Using a Drosophila melanogaster cell-free translation system, we found that this effect is mediated by the poly(A)-binding protein (PABP). miRNA repression was positively correlated with poly(A) tail length, but this stimulatory effect on repression was lost when translation was repressed by the tethered GW182 silencing domain rather than the miRNA-induced silencing complex (miRISC) itself. These findings are mechanistically explained by a notable function of PABP: it promotes association of miRISC with miRNA-regulated mRNAs. We also found that PABP association with mRNA rapidly diminished with miRISC recruitment and before detectable deadenylation. We integrated these data into a revised model for the function of PABP and the poly(A) tail in miRNA-mediated translational repression.
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Acknowledgements
We thank E. Izaurralde (Max Planck Institute for Developmental Biology, Tübingen, Germany) for GW182-SD-pAC5.1 and MBP-DmPABPC1 plasmids and for eIF4G antibody; C. Strein (EMBL, Heidelberg, Germany) for cloning and expression of Gst-λN-tagged GW182-SD; E. Izaurralde, L. Zekri and E. Huntzinger (Tübingen) for sharing unpublished results and their comments on this work; and all members of the Hentze laboratory, especially K. Duncan and J. Medenbach, for advice and discussions. This work was funded by a grant (FOR855 He 1442/13-2) from the Deutsche Forschungsgemeinschaft to M.W.H. C.K. is supported by an International Incoming Marie Curie fellowship (European Framework 7th, no. 253415).
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F.M., C.K., A.Z.-S. and M.W.H. designed experiments and analyzed results. F.M., C.K. and A.Z.-S. carried out experiments. F.M., C.K. and M.W.H. wrote the manuscript.
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Moretti, F., Kaiser, C., Zdanowicz-Specht, A. et al. PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding. Nat Struct Mol Biol 19, 603–608 (2012). https://doi.org/10.1038/nsmb.2309
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DOI: https://doi.org/10.1038/nsmb.2309
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