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.

  • Letter
  • Published:

Selective autophagy degrades DICER and AGO2 and regulates miRNA activity

Nature Cell Biology volume 14, pages 1314–1321 (2012)Cite this article

Subjects

A Corrigendum to this article was published on 31 July 2015

This article has been updated

Abstract

MicroRNAs (miRNAs) form a class of short RNAs (∼ 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined1,2 and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive3. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.

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: Levels of DICER and AGO proteins are regulated by autophagy.
Figure 2: DICER co-localizes and associates with autophagosomes.
Figure 3: DICER associates with NDP52 and is targeted to autophagy independently of an RNA cargo.
Figure 4: miRNA and miRNA* levels decrease when autophagy is inhibited for extended periods.
Figure 5: Autophagy is required for miRNA activity and engages post-transcriptional regulation of DICER.

Similar content being viewed by others

Change history

  • 10 July 2015

    In the version of this Letter originally published, the TUBA immunoblotting panel and the Coomassie-stained panel was used in Figure 5c (in the dataset corresponding to let-7 antagomir treatment, top set of panels), and reused in Figure 5e, without appropriate acknowledgement. The Coomassie-stained gel was vertically flipped in Figure 5e but the alignment of the lanes was maintained. The TUBA immunoblot and Coomassie-stained membranes represent experimental controls. The p62 panel in Figures 2j and 2k (CQ-treated) was also reused without appropriate attribution. In all cases of reuse of blots between panels, the samples were obtained within one representative experiment and processed in parallel. The authors confirm that all instances of vertical splicing of lanes, for example in Figs 1 and 3, were carried out in full compliance with the journal guidelines. All spliced samples were collected and processed in a single experiment. The original publication was missing Supplementary Fig. S3 containing the uncropped scans of the blots; this has now been included online.

References

  1. Kim, V. N., Han, J. & Siomi, M. C. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–39 (2009).

    Article  CAS  Google Scholar 

  2. Krol, J., Loedige, I. & Filipowicz, W. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Gene. 11, 597–610 (2010).

    Article  CAS  Google Scholar 

  3. Siomi, H. & Siomi, M. C. Post transcriptional regulation of microRNA biogenesis in animals. Mol. Cell 38, 323–32 (2010).

    Article  CAS  Google Scholar 

  4. Eulalio, A., Huntzinger, E. & Izaurralde, E. GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat. Struct. Mol. Biol. 15, 346–53 (2008).

    Article  CAS  Google Scholar 

  5. Bazzini, A. A., Lee, M. T. & Giraldez, A. J. Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336, 233–237 (2012).

    Article  CAS  Google Scholar 

  6. Ameres, S. L. et al. Target RNA-directed trimming and tailing of small silencing RNAs. Science 328, 1534–9 (2010).

    Article  CAS  Google Scholar 

  7. Chatterjee, S. & Grosshans, H. Active turnover modulates mature microRNA activity in Caenorhabditis elegans. Nature 461, 546–9 (2009).

    Article  CAS  Google Scholar 

  8. Johnston, M., Geoffroy, M. C., Sobala, A., Hay, R. & Hutvagner, G. HSP90 protein stabilizes unloaded argonaute complexes and microscopic P-bodies in human cells. Mol. Biol. Cell 21, 1462–9 (2010).

    Article  CAS  Google Scholar 

  9. Mayr, C. & Bartel, D. P. Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138, 673–84 (2009).

    Article  CAS  Google Scholar 

  10. Vaucheret, H., Vazquez, F., Crete, P. & Bartel, D. P. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187–97 (2004).

    Article  CAS  Google Scholar 

  11. Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147, 728–41 (2011).

    Article  CAS  Google Scholar 

  12. Pankiv, S. et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 282, 24131–45 (2007).

    Article  CAS  Google Scholar 

  13. Thurston, T. L., Wandel, M. P., von Muhlinen, N., Foeglein, A. & Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–8 (2012).

    Article  CAS  Google Scholar 

  14. Shaid, S., Brandts, C. H., Serve, H. & Dikic, I. Ubiquitination and selective autophagy. Cell Death Diff.http://dx.doi.org/10.1038 (2012).

  15. Watanabe, Y. & Tanaka, M. p62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate. J. Cell Sci. 124, 2692–701 (2011).

    Article  CAS  Google Scholar 

  16. Gibbings, D. J., Ciaudo, C., Erhardt, M. & Voinnet, O. Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat. Cell Biol. 11, 1143–9 (2009).

    Article  CAS  Google Scholar 

  17. Haase, A. D. et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 6, 961–7 (2005).

    Article  CAS  Google Scholar 

  18. Lee, Y. S. et al. Silencing by small RNAs is linked to endosomal trafficking. Nat. Cell Biol. 11, 1150–6 (2009).

    Article  CAS  Google Scholar 

  19. Gibbings, D. & Voinnet, O. Control of RNA silencing and localization by endolysosomes. Trends Cell Biol. 20, 491–501 (2010).

    Article  CAS  Google Scholar 

  20. Friend, K. et al. A conserved PUF-Ago-eEF1A complex attenuates translation elongation. Nat. Struct. Mol. Biol. 19, 176–83 (2012).

    Article  CAS  Google Scholar 

  21. Pare, J. M. et al. Hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and P-bodies. Mol. Biol. Cell 20, 3273–84 (2009).

    Article  CAS  Google Scholar 

  22. Liu, J., Valencia-Sanchez, M. A., Hannon, G. J. & Parker, R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, 719–23 (2005).

    Article  CAS  Google Scholar 

  23. Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–26 (2010).

    Article  CAS  Google Scholar 

  24. Marzella, L., Ahlberg, J. & Glaumann, H. Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization. J. Cell Biol. 93, 144–54 (1982).

    Article  CAS  Google Scholar 

  25. Di, Y. et al. HCC-associated protein HCAP1, a variant of GEMIN4, interacts with zinc-finger proteins. J. Biochem. 133, 713–8 (2003).

    Article  CAS  Google Scholar 

  26. Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–55 (2005).

    Article  CAS  Google Scholar 

  27. Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–8 (2002).

    Article  CAS  Google Scholar 

  28. Gantier, M. P. et al. Analysis of microRNA turnover in mammalian cells following Dicer1 ablation. Nucleic Acids Res. 39, 5692–703 (2011).

    Article  CAS  Google Scholar 

  29. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–4 (2005).

    Article  CAS  Google Scholar 

  30. Sakurai, K. et al. A role for human Dicer in pre-RISC loading of siRNAs. Nucleic Acids Res. 39, 1510–25 (2011).

    Article  CAS  Google Scholar 

  31. Noland, C. L., Ma, E. & Doudna, J. A. siRNA repositioning for guide strand selection by human Dicer complexes. Mol. Cell 43, 110–21 (2011).

    Article  CAS  Google Scholar 

  32. Iwasaki, S. et al. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol. Cell 39, 292–9 (2010).

    Article  CAS  Google Scholar 

  33. Mamane, Y., Petroulakis, E., LeBacquer, O. & Sonenberg, N. mTOR, translation initiation and cancer. Oncogene 25, 6416–22 (2006).

    Article  CAS  Google Scholar 

  34. Doench, J. G. & Sharp, P. A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–11 (2004).

    Article  CAS  Google Scholar 

  35. Slack, F. let-7 microRNA reduces tumor growth. Cell Cycle 8, 1823 (2009).

    Article  Google Scholar 

  36. Obad, S. et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat. Genet. 43, 371–8 (2011).

    Article  CAS  Google Scholar 

  37. Csorba, T., Lozsa, R., Hutvagner, G. & Burgyan, J. Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1. Plant J. Cell Mol. Biol. 62, 463–72 (2010).

    Article  CAS  Google Scholar 

  38. Derrien, B. et al. Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway. Proc. Natl Acad. Sci. USA 109, 15942–15946 (2012).

    Article  CAS  Google Scholar 

  39. Wander, S. A., Hennessy, B. T. & Slingerland, J. M. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J. Clin. Inv. 121, 1231–41 (2011).

    Article  CAS  Google Scholar 

  40. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–8 (2005).

    Article  CAS  Google Scholar 

  41. Rudel, S., Flatley, A., Weinmann, L., Kremmer, E. & Meister, G. A multifunctional human Argonaute2-specific monoclonal antibody. RNA 14, 1244–53 (2008).

    Article  Google Scholar 

  42. Eystathioy, T. et al. A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. Cell 13, 1338–51 (2002).

    Article  CAS  Google Scholar 

  43. Doench, J. G., Petersen, C. P. & Sharp, P. A. siRNAs can function as miRNAs. Gen. Dev. 17, 438–42 (2003).

    Article  CAS  Google Scholar 

  44. Klionsky, D. J., Elazar, Z., Seglen, P. O. & Rubinsztein, D. C. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4, 849–950 (2008).

    Article  CAS  Google Scholar 

  45. Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol. 7, 1267–74 (2005).

    Article  Google Scholar 

  46. Mesquita, F. S. et al. The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS Pathog. 8, e1002743 (2012).

    Article  CAS  Google Scholar 

  47. Costes, S. V. et al. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).

    Article  CAS  Google Scholar 

  48. Spiegelhalter, C. et al. From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy. PLoS ONE 5, e9014 (2010).

    Article  Google Scholar 

  49. Sahu, R. et al. Microautophagy of cytosolic proteins by late endosomes. Dev. Cell. 20, 131–9 (2011).

    Article  CAS  Google Scholar 

  50. Chi, S. W., Zang, J. B., Mele, A. & Darnell, R. B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–86 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank M. Johnston (ETH-Zurich, Switzerland) for providing HEK293T cells stably transfected with Tet-inducible Flag–AGO2 as well as the protocol for detection of ubiquitylated Flag–AGO2. Financial support was provided by a core grant from ETH-Z to O.V., and the Pasteur Institute to P.C. S.M. is a Wellcome Trust Research Career Development Fellow. The authors thank K. McGourty and D. Li for helpful discussions.

Author information

Author notes

  1. Derrick Gibbings and Serge Mostowy: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biology, Swiss Federal Institute of Technology (ETH-Z), Zürich 8092, Switzerland

    Derrick Gibbings, Florence Jay & Olivier Voinnet

  2. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa K1H 8M5, Canada

    Derrick Gibbings

  3. Département de Biologie Cellulaire et Infection, Institut Pasteur, Unité des Interactions Bactéries-Cellules, 75015 Paris, France

    Serge Mostowy & Pascale Cossart

  4. Inserm, Unité 604, 75015 Paris, France

    Serge Mostowy & Pascale Cossart

  5. INRA, USC2020, 75015 Paris, France

    Serge Mostowy & Pascale Cossart

  6. Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK

    Serge Mostowy

  7. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Institut National de la Santé et de la Recherche Médicale Unité 964, 1 rue Laurent Fries, 67404 Illkirch, France

    Yannick Schwab

  8. Institut de Biologie Moléculaire des Plantes (IBMP), 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France

    Olivier Voinnet

Authors
  1. Derrick Gibbings
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Serge Mostowy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Florence Jay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yannick Schwab
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pascale Cossart
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olivier Voinnet
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.G. conceived the hypothesis. D.G., S.M., F.J. and Y.S. performed and analysed experiments. D.G., S.M. and O.V. designed the overall research. D.G., S.M., P.C. and O.V. wrote the manuscript.

Corresponding authors

Correspondence to Derrick Gibbings, Serge Mostowy or Olivier Voinnet.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 362 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gibbings, D., Mostowy, S., Jay, F. et al. Selective autophagy degrades DICER and AGO2 and regulates miRNA activity. Nat Cell Biol 14, 1314–1321 (2012). https://doi.org/10.1038/ncb2611

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2611

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