title: Sumoylation of human argonaute 2 at lysine-402 regulates its stability.
abstract: Gene silencing by small RNAs has emerged as a powerful post-transcriptional regulator of gene expression, however processes underlying regulation of the small RNA pathway in vivo are still largely elusive. Here, we identified sumoylation as a novel post-translational modification acting on Ago2, the main effector of small RNA-mediated gene silencing. We demonstrate that Ago2 can be modified by SUMO1 and SUMO2/3 and identified Lys402 as the major Ago2 sumoylation site in vivo. Ago2 physically interacts with the SUMO E2 conjugating enzyme Ubc9 and the E3 ligase RanBP2 facilitates Ago2 sumoylation in vitro. Mutation of Lys402 enhances the stability of Ago2 protein and impairment of cellular sumoylation by siRNA- or shRNA-mediated extinction of Ubc9 or in Ubc9 knockout mouse tissues results in increased steady-state levels and enhanced stability of Ago2. Similarly, knockdown of RanBP2 or of the SAE2 E1 enzyme enhances Ago2 protein levels. Lys402 is located in the L2g1 loop linking the PAZ and PIWI domains of Ago2, in the immediate vicinity of Tyr393 which can be phosphorylated, implying that the L2g1 linker represents an easily accessible hot spot for post-translational modifications. Altogether, our results show that sumoylation of Ago2 at Lys402 negatively regulates its stability, thereby establishing a first link between SUMO and the small RNA machinery.
authors.affiliation: Laboratory of Nuclear Organization and Oncogenesis, Institut Pasteur, Paris, France; Institut National de la Santé et de la Recherche Médicale, U993, Paris, France; Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.
authors.forename: Umut
authors.initials: U
authors.lastname: Sahin
authors.affiliation: Laboratory of Nuclear Organization and Oncogenesis, Institut Pasteur, Paris, France; Institut National de la Santé et de la Recherche Médicale, U993, Paris, France; Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lapaquette
authors.affiliation: Laboratory of Nuclear Organization and Oncogenesis, Institut Pasteur, Paris, France; Institut National de la Santé et de la Recherche Médicale, U993, Paris, France; Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Andrieux
authors.affiliation: Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, UMR7590, Paris, France.
authors.forename: Guilhem
authors.initials: G
authors.lastname: Faure
authors.affiliation: Laboratory of Nuclear Organization and Oncogenesis, Institut Pasteur, Paris, France; Institut National de la Santé et de la Recherche Médicale, U993, Paris, France; Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Dejean
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D020868:Gene Silencing / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation / Q000235:genetics*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D034741:RNA, Small Interfering; D025842:SUMO-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins; C094493:ran-binding protein 2; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008239:Lysine