Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Kim, V. N. MicroRNA biogenesis: coordinated cropping and dicing. Nature Rev. Mol. Cell Biol. 6, 376–385 (2005)

    CAS  Article  Google Scholar 

  2. Gregory, R. I., Chendrimada, T. P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005)

    CAS  Article  Google Scholar 

  3. Jackson, R. J. & Standart, N. How do microRNAs regulate gene expression? Sci. STKE 2007, re1 (2007)

    Article  Google Scholar 

  4. Valencia-Sanchez, M. A., Liu, J., Hannon, G. J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006)

    CAS  Article  Google Scholar 

  5. Cook, H. A., Koppetsch, B. S., Wu, J. & Theurkauf, W. E. The Drosophila SDE3 homolog armitage is required for oskar mRNA silencing and embryonic axis specification. Cell 116, 817–829 (2004)

    CAS  Article  Google Scholar 

  6. Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841 (2004)

    CAS  Article  Google Scholar 

  7. Ceci, M. et al. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426, 579–584 (2003)

    ADS  CAS  Article  Google Scholar 

  8. Russell, D. W. & Spremulli, L. L. Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ. J. Biol. Chem. 254, 8796–8800 (1979)

    CAS  PubMed  Google Scholar 

  9. Valenzuela, D. M., Chaudhuri, A. & Maitra, U. Eukaryotic ribosomal subunit anti-association activity of calf liver is contained in a single polypeptide chain protein of Mr = 25,500 (eukaryotic initiation factor 6). J. Biol. Chem. 257, 7712–7719 (1982)

    CAS  PubMed  Google Scholar 

  10. Raychaudhuri, P., Stringer, E. A., Valenzuela, D. M. & Maitra, U. Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000). J. Biol. Chem. 259, 11930–11935 (1984)

    CAS  PubMed  Google Scholar 

  11. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005)

    ADS  CAS  Article  Google Scholar 

  12. Si, K., Chaudhuri, J., Chevesich, J. & Maitra, U. Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6. Proc. Natl Acad. Sci. USA 94, 14285–14290 (1997)

    ADS  CAS  Article  Google Scholar 

  13. Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005)

    CAS  Article  Google Scholar 

  14. Nelson, P. T., Hatzigeorgiou, A. G. & Mourelatos, Z. miRNP:mRNA association in polyribosomes in a human neuronal cell line. RNA 10, 387–394 (2004)

    CAS  Article  Google Scholar 

  15. Wu, L. & Belasco, J. G. Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. Mol. Cell. Biol. 25, 9198–9208 (2005)

    CAS  Article  Google Scholar 

  16. Basu, U., Si, K., Warner, J. R. & Maitra, U. The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis. Mol. Cell. Biol. 21, 1453–1462 (2001)

    CAS  Article  Google Scholar 

  17. Strezoska, Z., Pestov, D. G. & Lau, L. F. Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5.8S RRNA processing and 60S ribosome biogenesis. Mol. Cell. Biol. 20, 5516–5528 (2000)

    CAS  Article  Google Scholar 

  18. Si, K. & Maitra, U. The Saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation factor. Mol. Cell. Biol. 19, 1416–1426 (1999)

    CAS  Article  Google Scholar 

  19. Sanvito, F. et al. The B4 integrin interactor p27BBP/eIF6 is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly. J. Cell Biol. 14, 823–837 (1999)

    Article  Google Scholar 

  20. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005)

    CAS  Article  Google Scholar 

  21. Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854 (1993)

    CAS  Article  Google Scholar 

  22. Wightman, B., Ha, I. & Ruvkun, G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862 (1993)

    CAS  Article  Google Scholar 

  23. Moss, E. G., Lee, R. C. & Ambros, V. The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell 88, 637–646 (1997)

    CAS  Article  Google Scholar 

  24. 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)

    CAS  Article  Google Scholar 

  25. Arasu, P., Wightman, B. & Ruvkun, G. Temporal regulation of lin-14 by the antagonistic action of two other heterochronic genes, lin-4 and lin-28. Genes Dev. 5, 1825–1833 (1991)

    CAS  Article  Google Scholar 

  26. Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000)

    ADS  CAS  Article  Google Scholar 

  27. Feinbaum, R. & Ambros, V. The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. Dev. Biol. 210, 87–95 (1999)

    CAS  Article  Google Scholar 

  28. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001)

    CAS  Article  Google Scholar 

  29. Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W. & Sontheimer, E. J. A. Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94 (2004)

    CAS  Article  Google Scholar 

  30. Pillai, R. S. et al. Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309, 1573–1576 (2005)

    ADS  CAS  Article  Google Scholar 

  31. 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)

    ADS  CAS  Article  Google Scholar 

  32. 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)

    CAS  Article  Google Scholar 

  33. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003)

    ADS  CAS  Article  Google Scholar 

  34. Reinhart, B. J. & Ruvkun, G. Isoform-specific mutations in the Caenorhabditis elegans heterochronic gene lin-14 affect stage-specific patterning. Genetics 157, 199–209 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kiriakidou, M. et al. A combined computational-experimental approach predicts human microRNA targets. Genes Dev. 18, 1165–1178 (2004)

    CAS  Article  Google Scholar 

  36. Ji, X., Kong, J. & Liebhaber, S. A. In vivo association of the stability control protein αCP with actively translating mRNAs. Mol. Cell. Biol. 23, 899–907 (2003)

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Amy E. Pasquinelli or Ramin Shiekhattar.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-6 with Legends and Supplementary Table 1. (PDF 1000 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05841

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing