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Homeostatic control of Argonaute stability by microRNA availability

Nature Structural & Molecular Biology volume 20, pages 789–795 (2013)Cite this article

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Abstract

Homeostatic mechanisms regulate the abundance of several components in small-RNA pathways. We used Drosophila and mammalian systems to demonstrate a conserved homeostatic system in which the status of miRNA biogenesis controls Argonaute protein stability. Clonal analyses of multiple mutants of core Drosophila miRNA factors revealed that stability of the miRNA effector AGO1 is dependent on miRNA biogenesis. Reciprocally, ectopic transcription of miRNAs within in vivo clones induced accumulation of AGO1, as did genetic interference with the ubiquitin-proteasome system. In mouse cells, we found that the stability of Ago2 declined in Dicer-knockout cells and was rescued by proteasome blockade or introduction of either Dicer plasmid or Dicer-independent miRNA constructs. Notably, Dicer-dependent miRNA constructs generated pre-miRNAs that bound Ago2 but did not rescue Ago2 stability. We conclude that Argonaute levels are finely tuned by cellular availability of mature miRNAs and the ubiquitin-proteasome system.

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Figure 1: Drosophila AGO1 protein level is reduced in the absence of miRNA biogenesis and accumulates upon depletion of GW182.
Figure 2: miRNA biogenesis stabilizes AGO1 post-transcriptionally, but siRNA biogenesis does not stabilize AGO2 in Drosophila.
Figure 3: Drosophila AGO1 accumulates with increased miRNA transcription and is turned over by the ubiquitin-proteasome system.
Figure 4: Mammalian Ago2 is stabilized by ongoing miRNA biogenesis.
Figure 5: Accumulation of mouse and Drosophila Argonautes is dependent on loading with functional small RNAs.

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Acknowledgements

We thank G. Meister (Universität Regensburg, Regensburg, Germany), M. Overholtzer (Sloan-Kettering Institute, New York, New York, USA), J. Liu (Sloan-Kettering Institute, New York, New York, USA), X. Jiang (Sloan-Kettering Institute, New York, New York, USA), K. Okamura (Temasek Institute, Singapore), V. Kim (Seoul National University, Seoul, Korea), H. Siomi (Keio University, Tokyo, Japan), A. Tarakhovsky (Rockefeller University, New York, New York, USA), D. O'Carroll (European Molecular Biology Laboratory, Monterotondo Scalo, Italy), R. Carthew (Northwestern University, Chicago, Illinois, USA), F. Schweisguth (Institut Pasteur, Paris, France), F. Gao (University of Massachusetts Medical School, Worcester, Massachusetts, USA), Paul Lasko (McGill University, Montreal, Canada), the Vienna Drosophila RNAi Center (Vienna, Austria), the Transgenic RNAi Project at Harvard Medical School (Boston, Massachusetts, USA) and the Bloomington Drosophila Stock Center (Bloomington, Indiana, USA) for reagents and discussions. Work in J.-L.L.'s group was supported by the UK Medical Research Council. G.A. was supported by the Malaysian Ministry of Higher Education. Work in E.C.L.'s group was supported by the US National Institutes of Health R01-GM083300.

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Author notes

  1. Ghows Azzam

    Present address: Present address: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.,

  2. Peter Smibert, Jr-Shiuan Yang and Ghows Azzam: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Developmental Biology, Sloan-Kettering Institute, New York, New York, USA

    Peter Smibert, Jr-Shiuan Yang & Eric C Lai

  2. Department of Physiology, Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford, Oxford, UK

    Ghows Azzam & Ji-Long Liu

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Contributions

P.S., G.A. and J.-L.L. made the initial observations that demonstrated AGO1 homeostasis in vivo. Mechanistic experiments were carried out in flies by P.S., in S2 cells by P.S. and J.-S.Y. and in mammalian cells by J.-S.Y. P.S., J.-S.Y. and E.C.L. wrote the text with input from G.A. and J.-L.L.

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Correspondence to Ji-Long Liu or Eric C Lai.

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Smibert, P., Yang, JS., Azzam, G. et al. Homeostatic control of Argonaute stability by microRNA availability. Nat Struct Mol Biol 20, 789–795 (2013). https://doi.org/10.1038/nsmb.2606

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