MicroRNAs (miRs) inhibit protein synthesis by mechanisms that are as yet unresolved1,2,3,4,5,6,7,8,9,10,11. We developed a cell-free system from Drosophila melanogaster embryos that faithfully recapitulates miR2-mediated translational control by means of the 3′ untranslated region of the D. melanogaster reaper messenger RNA. Here we show that miR2 inhibits translation initiation without affecting mRNA stability. Surprisingly, miR2 induces the formation of dense (heavier than 80S) miRNPs (‘pseudo-polysomes’) even when polyribosome formation and 60S ribosomal subunit joining are blocked. An mRNA bearing an ApppG instead of an m7GpppG cap structure escapes the miR2-mediated translational block. These results directly show the inhibition of m7GpppG cap-mediated translation initiation as the mechanism of miR2 function, and uncover pseudo-polysomal messenger ribonucleoprotein assemblies that may help to explain earlier findings.
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Petersen, C. P., Bordeleau, M. E., Pelletier, J. & Sharp, P. A. Short RNAs repress translation after initiation in mammalian cells. Mol. Cell 21, 533–542 (2006)
Nottrott, S., Simard, M. J. & Richter, J. D. Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nature Struct. Mol. Biol. 13, 1108–1114 (2006)
Maroney, P. A., Yu, Y., Fisher, J. & Nilsen, T. W. Evidence that microRNAs are associated with translating messenger RNAs in human cells. Nature Struct. Mol. Biol. 13, 1102–1107 (2006)
Olsen, P. H. & Ambros, V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216, 671–680 (1999)
Seggerson, K., Tang, L. & Moss, E. G. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev. Biol. 243, 215–225 (2002)
Pillai, R. S. et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573–1576 (2005)
Humphreys, D. T., Westman, B. J., Martin, D. I. & Preiss, T. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc. Natl Acad. Sci. USA 102, 16961–16966 (2005)
Wu, L., Fan, J. & Belasco, J. G. MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl Acad. Sci. USA 103, 4034–4039 (2006)
Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005)
Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004)
Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004)
Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004)
Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005)
Xie, X. et al. Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434, 338–345 (2005)
Lim, L. P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005)
Yekta, S., Shih, I. H. & Bartel, D. P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004)
Sen, G. L. & Blau, H. M. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nature Cell Biol. 7, 633–636 (2005)
Liu, J., Valencia-Sanchez, M. A., Hannon, G. J. & Parker, R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nature Cell Biol. 7, 719–723 (2005)
Stark, A., Brennecke, J., Russell, R. B. & Cohen, S. M. Identification of Drosophila MicroRNA targets. PLoS Biol. 1, 397–409 (2003)
Leaman, D. et al. Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121, 1097–1108 (2005)
Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 (2001)
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191–3197 (1999)
Lecellier, C. H. et al. A cellular microRNA mediates antiviral defense in human cells. Science 308, 557–560 (2005)
Gray, N. K. & Hentze, M. W. Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J. 13, 3882–3891 (1994)
Gebauer, F., Grskovic, M. & Hentze, M. W. Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA. Mol. Cell 11, 1397–1404 (2003)
Hershey, J. W. & Monro, R. E. A competitive inhibitor of the GTP reaction in protein synthesis. J. Mol. Biol. 18, 68–76 (1966)
Anthony, D. D. & Merrick, W. C. Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J. Biol. Chem. 267, 1554–1562 (1992)
Beckmann, K., Grskovic, M., Gebauer, F. & Hentze, M. W. A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila. Cell 122, 529–540 (2005)
Chekulaeva, M., Hentze, M. W. & Ephrussi, A. Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124, 521–533 (2006)
Thoma, C. et al. Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP. Mol. Cell 15, 925–935 (2004)
Preiss, T., Muckenthaler, M. & Hentze, M. W. Poly(A)-tail-promoted translation in yeast: implications for translational control. RNA 4, 1321–1331 (1998)
Ostareck, D. H. et al. Lipoxygenase mRNA silencing in erythroid differentiation: The 3′UTR regulatory complex controls 60S ribosomal subunit joining. Cell 104, 281–290 (2001)
Valoczi, A. et al. Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 32, e175 (2004)
We thank S. Cohen and J. Brennecke for advice, and R. Jackson and the members of the Hentze laboratory for discussions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.W.H.
Author Contributions R.T. performed the experiments. R.T. and M.W.H. designed, analysed and interpreted the experiments and wrote the paper.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Thermann, R., Hentze, M. Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875–878 (2007). https://doi.org/10.1038/nature05878
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