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.

  • Article
  • Published:

PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding

Nature Structural & Molecular Biology volume 19, pages 603–608 (2012)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Depletion of PABP abrogates poly(A) tail stimulation of miRNA-mediated repression.
Figure 2: The poly(A) tail stimulates repression by endogenous miR2 but not by tethered GW182-SD.
Figure 3: PABP is displaced from repressed mRNAs early during miRNA-mediated translational repression.
Figure 4: PABP and the poly(A) tail enhance AGO1 association with target mRNAs.
Figure 5: PABP displacement precedes mRNA deadenylation.

Similar content being viewed by others

References

  1. Bushati, N. & Cohen, S.M. microRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).

    Article  CAS  Google Scholar 

  2. Huntzinger, E. & Izaurralde, E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat. Rev. Genet. 12, 99–110 (2011).

    Article  CAS  Google Scholar 

  3. Kahvejian, A., Roy, G. & Sonenberg, N. The mRNA closed-loop model: the function of PABP and PABP-interacting proteins in mRNA translation. Cold Spring Harb. Symp. Quant. Biol. 66, 293–300 (2001).

    Article  CAS  Google Scholar 

  4. Fabian, M.R. et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol. Cell 35, 868–880 (2009).

    Article  CAS  Google Scholar 

  5. Mishima, Y. et al. Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish. Proc. Natl. Acad. Sci. USA 109, 1104–1109 (2012).

    Article  CAS  Google Scholar 

  6. Zekri, L., Huntzinger, E., Heimstädt, S. & Izaurralde, E. The silencing domain of GW182 interacts with PABC1 to promote translational repression and degradation of miRNA targets and is required for target release. Mol. Cell. Biol. 29, 6220–6231 (2009).

    Article  CAS  Google Scholar 

  7. Huntzinger, E., Braun, J.E., Heimstädt, S., Zekri, L. & Izaurralde, E. Two PABC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing. EMBO J. 29, 4146–4160 (2010).

    Article  CAS  Google Scholar 

  8. Jinek, M., Fabian, M.R., Coyle, A.M., Sonenberg, N. & Doudna, J. Structural insights into the human GW182-PABP interaction in microRNA-mediated deadenylation. Nat. Struct. Mol. Biol. 17, 238–240 (2010).

    Article  CAS  Google Scholar 

  9. Kozlov, G., Safaee, N., Rosenauer, A. & Gehring, K. Structural basis of binding of P-body-associated proteins GW182 and Ataxin-2 by the Mlle domain of poly(A)-binding protein. J. Biol. Chem. 285, 13599–13606 (2010).

    Article  CAS  Google Scholar 

  10. Eulalio, A., Tritschler, F. & Izaurralde, E. The GW182 protein family in animal cells: New insights into domains required for miR-mediated gene silencing. RNA 15, 1433–1442 (2009).

    Article  CAS  Google Scholar 

  11. Braun, J.E., Huntzinger, E., Fauser, M. & Izaurralde, E. GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets. Mol. Cell 44, 120–133 (2011).

    Article  CAS  Google Scholar 

  12. Eulalio, A. et al. Deadenylation is a widespread effect of miRNA regulation. RNA 15, 21–32 (2009).

    Article  CAS  Google Scholar 

  13. Fukaya, T. & Tomari, Y. PABP is not essential for microRNA-mediated translational repression and deadenylation in vitro. EMBO J. 30, 4998–5009 (2011).

    Article  CAS  Google Scholar 

  14. Wu, L., Fan, J. & Belasco, J.G. MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. USA 103, 4034–4039 (2006).

    Article  CAS  Google Scholar 

  15. Zdanowicz, A. et al. D. melanogaster miR2 primarily targets the m7GpppN cap structure for translational repression. Mol. Cell 35, 881–888 (2009).

    Article  CAS  Google Scholar 

  16. Iwasaki, S., Kawamata, T. & Tomari, Y. D. melanogaster argonaute1 and argonaute2 employ distinct mechanisms for translational repression. Mol. Cell 34, 58–67 (2009).

    Article  CAS  Google Scholar 

  17. Beilharz, T.H. et al. microRNA-mediated messenger RNA deadenylation contributes to translational repression in mammalian cells. PLoS ONE 4, e6783 (2009).

    Article  Google Scholar 

  18. Ricci, E.P. et al. Activation of a microRNA response in trans reveals a new role for poly(A) in translational repression. Nucleic Acids Res. 39, 5215–5231 (2011).

    Article  CAS  Google Scholar 

  19. Thangima Zannat, M., Bhattacharjee, R.B. & Bag, J. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells. Biochem. Biophys. Res. Commun. 408, 375–381 (2011).

    Article  Google Scholar 

  20. Blagden, S.P. et al. D. melanogaster Larp associates with poly(A)-binding protein and is required for male fertility and syncytial embryo development. Dev. Biol. 334, 186–197 (2009).

    Article  CAS  Google Scholar 

  21. Kahvejian, A., Svitkin, Y.V., Sukarieh, R., M'Boutchou, M.N. & Sonenberg, N. Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes Dev. 19, 104–113 (2005).

    Article  CAS  Google Scholar 

  22. Thermann, R. & Hentze, M.W. D. melanogaster miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875–878 (2007).

    Article  CAS  Google Scholar 

  23. Till, S. et al. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat. Struct. Mol. Biol. 14, 897–903 (2007).

    Article  CAS  Google Scholar 

  24. Svitkin, Y.V. & Sonenberg, N. An efficient system for cap- and poly(A)-dependent translation in vitro. Methods Mol. Biol. 257, 155–170 (2004).

    CAS  PubMed  Google Scholar 

  25. Moretti, F., Thermann, R. & Hentze, M.W. Mechanism of translational regulation by miR-2 from sites in the 5′ untranslated region or the open reading frame. RNA 16, 2493–2502 (2010).

    Article  CAS  Google Scholar 

  26. Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    Article  CAS  Google Scholar 

  27. Eulalio, A. et al. The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing. Nucleic Acids Res. 37, 2974–2983 (2009).

    Article  CAS  Google Scholar 

  28. Chekulaeva, M., Filipowicz, W. & Parker, R. Multiple independent domains of dGW182 function in miRNA-mediated repression in D. melanogaster. RNA 15, 794–803 (2009).

    Article  CAS  Google Scholar 

  29. Tan, R. & Frankel, A.D. Structural variety of arginine-rich RNA-binding peptides. Proc. Natl. Acad. Sci. USA 92, 5282–5286 (1995).

    Article  CAS  Google Scholar 

  30. Tomari, Y., Du, T. & Zamore, P.D. Sorting of D. melanogaster small silencing RNAs. Cell 130, 299–308 (2007).

    Article  CAS  Google Scholar 

  31. Stark, A., Brennecke, J., Russel, R.B. & Cohen, S.M. Identification of D. melanogaster microRNA targets. PLoS Biol. 1, e60 (2003).

    Article  Google Scholar 

  32. Chekulaeva, M. et al. miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nat. Struct. Mol. Biol. 18, 1218–1226 (2011).

    Article  CAS  Google Scholar 

  33. Simón, E. & Seraphin, B. A specific role for the C-terminal region of the poly(A)-binding protein in mRNA decay. Nucleic Acids Res. 35, 6017–6028 (2007).

    Article  Google Scholar 

  34. Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D. & Parker, R. Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J. 21, 1427–1436 (2002).

    Article  CAS  Google Scholar 

  35. Fabian, M.R. et al. miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT. Nat. Struct. Mol. Biol. 18, 1211–1217 (2011).

    Article  CAS  Google Scholar 

  36. Kim, H.H. et al. HuR recruits let-7/RISC to repress c-Myc expression. Genes Dev. 23, 1743–1748 (2009).

    Article  CAS  Google Scholar 

  37. Grskovic, M., Hentze, M.W. & Gebauer, F. A co-repressor assembly nucleated by Sex-lethal in the 3′UTR mediates translational control of D. melanogaster msl-2 mRNA. EMBO J. 22, 5571–5581 (2003).

    Article  CAS  Google Scholar 

  38. Thoma, C., Fraterman, S., Gentzel, M., Wilm, M. & Hentze, M.W. Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail. RNA 14, 1579–1589 (2008).

    Article  CAS  Google Scholar 

  39. Czaplinski, K. et al. Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-β-related mRNA during oogenesis. Dev. Cell 8, 505–515 (2005).

    Article  CAS  Google Scholar 

Download references

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).

Author information

Author notes

  1. Constanze Kaiser & Agnieszka Zdanowicz-Specht

    Present address: Present addresses: Roche Pharma (Schweiz), Reinach, Switzerland (C.K.); Roche Pharma, Grenzach-Wyhlen, Germany (A.Z.-S.).,

  2. Francesca Moretti and Constanze Kaiser: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Heidelberg, Germany

    Francesca Moretti, Constanze Kaiser, Agnieszka Zdanowicz-Specht & Matthias W Hentze

Authors
  1. Francesca Moretti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Constanze Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Agnieszka Zdanowicz-Specht
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthias W Hentze
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Matthias W Hentze.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 4353 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2309

This article is cited by

Search

Advanced 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