MicroRNA silencing through RISC recruitment of eIF6

Abstract

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10—which is the homologue of Drosophila translational repressor Armitage—and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

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Figure 1: Biochemical and functional analysis of eIF6 in the miRNA pathway.
Figure 2: Impact of eIF6 depletion on let-7-responsive reporter mRNA levels and on cellular polysome profiles.
Figure 3: Depletion of eif-6 disrupts regulation of protein levels of lin-4 miRNA endogenous targets.
Figure 4: RNAi of eif-6 results in misregulation of lin-14 and lin-28 target mRNA levels.
Figure 5: Depletion of EIF-6 during C. elegans larval development.

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Acknowledgements

We thank Z. Mourelatos for providing the firefly luciferase plasmids containing let-7b binding sites; J. Belasco for Luc-lin28 (firefly luciferase reporter fused to the 2.7-kb 3′ UTR of human LIN28) and its mutants; G. Ruvkun and E. Moss for LIN-14 and LIN-28 antibodies, respectively; and J. Bracht, M. G. Lee, S. Bagga and G. Harris for technical assistance. We also thank T. Beer of the Wistar Proteomics Facility for expertise in the microcapillary HPLC/mass spectrometry. This work was supported by NIH (A.E.P., R.S., S.A.L.), the Searle and V Foundations (AEP), the Mathers Foundation (RS) and a Cooley’s Anemia Foundation Fellowship to X.J.

Author Contributions T.P.C., K.J.F. and X.J. contributed equally to the work. T.P.C. performed experiments in human cells with help from R.I.G. and D.B. X.J. performed Fig. 2 experiments. R.S., A.E.P. and S.A.L. wrote the manuscript.

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Correspondence to Amy E. Pasquinelli or Ramin Shiekhattar.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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Chendrimada, T., Finn, K., Ji, X. et al. MicroRNA silencing through RISC recruitment of eIF6. Nature 447, 823–828 (2007). https://doi.org/10.1038/nature05841

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