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Argonaute proteins couple chromatin silencing to alternative splicing

Nature Structural & Molecular Biology volume 19, pages 998–1004 (2012)Cite this article

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Abstract

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.

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Figure 1: Characterization of AGO.ca complexes.
Figure 2: The AGO2.ca complex is enriched in sRNAs close to splice junctions.
Figure 3: AGO1 and AGO2 regulate alternative splicing of CD44 transcripts.
Figure 4: AGO1 and AGO2 proteins are recruited to the coding region of the CD44 gene.
Figure 5: AGO1 and AGO2 bind to CD44 pre-mRNA.
Figure 6: Deposition of H3K9me3 and reduction of RNAP II elongation rate require AGO1 and AGO2.
Figure 7: sRNAs bound to the AGO2.ca complex are located on the CD44 gene.

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Acknowledgements

The authors thank A. Krainer (Cold Spring Harbor Laboratory) for the antibody to SRSF1 (anti-SRSF1) G. Meister (Regensburg University) for anti-AGO1 no. 4B8 and anti-AGO2 no. 11A9, Z. Mourelatos (University of Pennsylvania) for anti-AGO2 no. 2A8, A. Tarakhovsky (Rockefeller University), G. Hannon (Cold Spring Harbor Laboratory) and M. Otsuka (University of Tokyo) for the kind gift of knockout MEFs, A. Polesskaya (Centre National de la Recherche Scientifique) for generating the tagged AGO2 C2C12 cells, M. Zavolan and M. Khorshid (Basel University) for their help with bioinformatics, the Taplin Biological Mass Spectrometry Facility at Harvard Medical School for MS analysis, P. de la Grange (GenoSplice) for help with bioinformatics and J.B. Weitzman (University Paris Diderot), E. Allemand (Institut Pasteur) and L. Pritchard (Centre National de la Recherche Scientifique) for critical reading of the manuscript. This work was supported by the European Commission Sixth Framework Programme (Integrated Project Silencing RNAs: Organisers and Coordinators of Complexity in Eukaryotic Organisms (SIROCCO) contract number LSHG-CT-2006-037900, to A.H.-B.) and by the Agence Nationale de la Recherche (contract number ANR-11-BSV8-0013 to C.M., J.-C.A. and A.H.-B.).

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Author notes

  1. Robert Young

    Present address: Present address: Department of Mathematics, University of Toronto, Toronto, Ontario, Canada.,

  2. Christian Muchardt, Eric Batsché and Annick Harel-Bellan: These authors contributed equally to this work.

Authors and Affiliations

  1. Université Paris Sud, Laboratoire Epigenetique et Cancer, Formation de Recherche en Evolution, Gif-Sur-Yvette, France

    Maya Ameyar-Zazoua, Mouloud Souidi, Nadya Morozova, Jacques Mathieu & Annick Harel-Bellan

  2. Centre National de la Recherche Scientifique, Gif-Sur-Yvette, France

    Maya Ameyar-Zazoua, Mouloud Souidi, Nadya Morozova, Jacques Mathieu & Annick Harel-Bellan

  3. Commissariat à l'Energie Atomique, Saclay, Gif-sur-Yvette, France.,

    Maya Ameyar-Zazoua, Mouloud Souidi, Nadya Morozova & Annick Harel-Bellan

  4. Institut Pasteur, Unité de Régulation Epigénétique, Centre National de la Recherche Scientifique, Unité de Recherche Associée, Paris, France

    Maya Ameyar-Zazoua, Christophe Rachez, Christian Muchardt & Eric Batsché

  5. Epigénétique & Destin Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche, Université Paris Diderot, Sorbonne Paris Cité, Paris

    Philippe Robin, Lauriane Fritsch & Slimane Ait-Si-Ali

  6. Institut des Hautes Études Scientifiques, Bures-sur-Yvette, France

    Robert Young

  7. Centre d'Immunologie de Marseille-Luminy, Université d'Aix-Marseille, Marseille, France

    Romain Fenouil, Nicolas Descostes & Jean-Christophe Andrau

  8. Institut National de la Santé et de la Recherche Médicale, Marseille, France

    Romain Fenouil, Nicolas Descostes & Jean-Christophe Andrau

  9. Centre National de la Recherche Scientifique, Unité Mixte de Recherche, Marseille, France

    Romain Fenouil, Nicolas Descostes & Jean-Christophe Andrau

  10. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, Strasbourg, France.,

    Ali Hamiche

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Contributions

M.A.-Z. and M.S. conducted the biochemical characterization of AGO complexes; P.R. participated in mass-spectrometry analysis; M.A.-Z. and E.B. conducted functional splicing analyses and ChIP experiments; M.A.-Z. E.B. and C.R. conducted RNA-nChIP experiments; R.Y., E.B., N.M., R.F., N.D. and J.-C.A. analyzed RNA deep-sequencing data; E.B. analyzed exon arrays; J.M. conducted some of the RT-qPCRs; L.F. and S.A.-S.-A. contributed the analysis of AGO-H3K9 KMT interaction; A.H. proposed the tandem affinity purification–tagging procedure on chromatin and participated in the design of that part of the study; E.B. and C.M. initiated and supervised splicing analyses; A.H.-B. initiated and supervised AGO complex characterization; C.R. and S.A.-S.-A. participated in writing the paper; M.A.-Z., E.B., C.M. and A.H.-B. wrote the paper.

Corresponding authors

Correspondence to Eric Batsché or Annick Harel-Bellan.

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Ameyar-Zazoua, M., Rachez, C., Souidi, M. et al. Argonaute proteins couple chromatin silencing to alternative splicing. Nat Struct Mol Biol 19, 998–1004 (2012). https://doi.org/10.1038/nsmb.2373

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