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Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity

Nature Cell Biology volume 11, pages 1143–1149 (2009)Cite this article

An Erratum to this article was published on 01 October 2009

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Abstract

In animals, P-bodies or GW-bodies appear to cause the congregation of proteins involved in microRNA (miRNA)-mediated post-transcriptional silencing. The localization of P-bodies does not overlap with that of known organelles and are thus considered independent of lipid bilayers. Nonetheless, an miRNA effector protein, argonaute 2 (AGO2), was initially identified as membrane-associated, and some miRNAs have been found in secreted vesicles (exosomes) that derive from endo-lysosomal compartments called multivesicular bodies (MVBs). Proteins can be sorted in a ubiquitin-dependent manner into MVBs by three heteromeric subcomplexes, collectively termed ESCRT (endosomal sorting complex required for transport), to be further secreted in exosomes and/or degraded by the lysosome. Here we show that GW-bodies containing GW182 and AGO2, two main components of the RNA-induced silencing complex (RISC), are distinct from P-bodies due to their congregation with endosomes and MVBs. Moreover, miRNAs and miRNA-repressible mRNAs are enriched at these cellular membranes, suggesting that endosomes and/or MVBs are sites of miRNA-loaded RISC (miRISC) accumulation and, possibly, action. We further show that purified exosome-like vesicles secreted by MVBs are considerably enriched in GW182, but not P-body components, AGO2 or miRNA-repressible mRNA. Moreover, cells depleted of some ESCRT components show compromised miRNA-mediated gene silencing and over-accumulate GW182, which associates with ubiquitylated proteins. Therefore, GW182, possibly in association with a fraction of miRNA-loaded AGO2, is sorted into MVBs for secretion and/or lysosomal degradation. We propose that this process promotes continuous assembly or disassembly of membrane-associated miRISCs, which is possibly required for miRNA loading or target recognition and subsequent silencing.

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Figure 1: Purified exosomes contain mature miRNA and are enriched in GW182, but not DCP1A or Ge-1.
Figure 2: GW182, AGO2 and miRNA partition in continuous density gradients with endosomes and multivesicular bodies.
Figure 3: GW182, but not DCP1A, colocalizes with MVBs.
Figure 4: Endogenous GW182-positive/DCP1A-negative foci coincide with endosomes and MVBs more frequently than DCP1A-positive foci.
Figure 5: Knockdown of ESCRT complex components compromises miRNA activities.

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Change history

  • 08 September 2009

    In the version of this article initially published, the Merge column of Fig. 4 was incorrect. This error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This research was funded by a prize from the Schlumberger Foundation for Education and Research awarded to O.V. who is also supported by a starting grant from the European Research Council 'Frontiers of RNAi' ERC 210890. D.G. is supported by a postdoctoral fellowship from La Ligue Contre le Cancer. C. C. was supported by a postdoctoral fellowship from the Fondation pour la Recherche Médicale (FRM). The authors thank S. Amigorena, C. Thery and M. Ostrosowski for providing exosomes from HeLa and dendritic cells and for helpful comments; B. Lorber for help with dynamic light scattering; V. Cognat for bioinformatics analysis; G. Suffert and S. Pfeffer for providing miR206, miRK12-4 and estrogen receptor 3′UTR constructs, and P. Leblanc for independent preparations of density gradients.

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Authors and Affiliations

  1. IBMP-CNRS, UPR2357 Université de Strasbourg, 12 rue du Général Zimmer, Strasbourg Cedex, 67084, France

    Derrick J. Gibbings, Constance Ciaudo, Mathieu Erhardt & Olivier Voinnet

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Contributions

C.C. made small RNA libraries for 454 sequencing and designed and performed qRT-PCR studies. M.E. designed and performed electron microscopy experments. D.G. designed and performed all other experiments and helped write the manuscript. O.V. designed experiments and helped write the manuscript.

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Correspondence to Olivier Voinnet.

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Gibbings, D., Ciaudo, C., Erhardt, M. et al. Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat Cell Biol 11, 1143–1149 (2009). https://doi.org/10.1038/ncb1929

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