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Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes

Nature Structural & Molecular Biology volume 19, pages 517–524 (2012)Cite this article

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Abstract

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrPC (or PRNP) has poorly understood physiological functions. Whereas most PrPC is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrPC that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrPC variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrPC contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrPC preferentially binds AGO, and PrPC promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrPC. We propose that dynamic interactions between PrPC-enriched endosomes and subcellular foci of AGO underpin these effects.

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Figure 1: Ago preferentially interacts with transmembrane PrPC using GW/WG-motifs.
Figure 2: Mouse PrPC forms a complex with Ago2 in intact cells on MVB-like organelles.
Figure 3: PrPC associates with TNRC6 and DICER but does not affect pre-miRNA processing.
Figure 4: PrPC promotes assembly or stability of AGO complexes containing TNRC6 and miRNA-repressed mRNA.
Figure 5: PrPC promotes interaction of AGO with MVB.

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Acknowledgements

This work was supported by La Ligue Contre le Cancer (D.G., fellowship), Agence Nationale de la Recherche ANR-08-MIE-010 EXOPRION (P.L.), the Prix Liliane Bettencourt pour les Sciences du Vivant (OV) and ANR-08-BLAN-0206-01 'Ago-hook' (O.V. and T.L.). Rabbit antibody XN-1 was a gift from A. Aguzzi (University of Zurich).

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Author notes

  1. Derrick Gibbings, Pascal Leblanc, Florence Jay & Olivier Voinnet

    Present address: Present Addresses: Swiss Federal Institute of Technology, Department of Biology, Zurich, Switzerland (D.G., F.J. and O.V.); Centre National de la Recherche Scientifique, Laboratory of Molecular Biology of the Cell, Neuromuscular Differentiation Group, Ecole Normale Supérieure, Lyon, France (P.L.).,

  2. Pascal Leblanc and Florence Jay: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Strasbourg, France

    Derrick Gibbings, Florence Jay, Fabrice Michel & Olivier Voinnet

  2. Institut National de la Santé et de la Recherche Médicale, Laboratoire de Virologie Humain, Ecole Normale Supérieure, Lyon, France

    Pascal Leblanc & Sandrine Alais

  3. Centre National de la Recherche Scientifique, Laboratoire Génome et Développement des Plantes, Perpignan, France

    Dominique Pontier & Thierry Lagrange

  4. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

    Yannick Schwab

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Contributions

Y.S. did the electron microscopy experiments. D.P. and T.L. designed and carried out experiments defining the GW/WG-motif of PrPC. F.M. carried out the DICER assays. F.J. carried out some immunoprecipitations, western blots, FRET and all qRT-PCR. S.A. and P.L. generated cell lines stably expressing PrPC shRNA and prepared exosomes. D.G. designed experiments, wrote the manuscript and carried out immunoprecipitations, western blots, RNA extractions, confocal microscopy and analysis, density gradients, FRET, miRNA reporter assays and analysis of CtmPrPC. O.V. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Olivier Voinnet.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Methods (PDF 1877 kb)

Supplementary Video 1

Apparent interactions between MVB and foci of GFP-TNRC6A. U251MG expressing GFP–TNRC6A and loaded with the MVB–marker lipid NRhPE (red). (MOV 6241 kb)

Supplementary Video 2

Apparent interactions between MVB and foci of GFP-AGO2. U251MG expressing GFP–AGO2 and loaded with MVB–marker lipid NRhPE (red). (MOV 8166 kb)

Supplementary Video 3

Apparent interactions between MVB and foci of GFP-AGO2. HeLa expressing GFP–AGO2 and loaded with MVB–marker lipid NRhPE (red). (MOV 13585 kb)

Supplementary Video 4

Apparent interactions between PrPC + organelles and foci of GFP-TNRC6A. HeLa co–expressing GFP–AGO2 and Orange–PrPC (red). (MOV 5649 kb)

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Gibbings, D., Leblanc, P., Jay, F. et al. Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes. Nat Struct Mol Biol 19, 517–524 (2012). https://doi.org/10.1038/nsmb.2273

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