Sumoylation of human argonaute 2 at lysine-402 regulates its stability

Umut Sahin; Pierre Lapaquette; Alexandra Andrieux; Guilhem Faure; Anne Dejean

doi:10.1371/journal.pone.0102957

Sumoylation of human argonaute 2 at lysine-402 regulates its stability

PLoS One. 2014 Jul 18;9(7):e102957. doi: 10.1371/journal.pone.0102957. eCollection 2014.

Authors

Umut Sahin¹, Pierre Lapaquette¹, Alexandra Andrieux¹, Guilhem Faure², Anne Dejean¹

Affiliations

¹ 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.
² Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, UMR7590, Paris, France.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Argonaute Proteins / genetics
Argonaute Proteins / metabolism*
Cell Line, Tumor
Gene Silencing / physiology
HeLa Cells
Humans
Lysine / genetics
Lysine / metabolism*
Mice
Molecular Chaperones / genetics
Molecular Chaperones / metabolism
Nuclear Pore Complex Proteins / genetics
Nuclear Pore Complex Proteins / metabolism
Phosphorylation / genetics
Protein Processing, Post-Translational / genetics
RNA, Small Interfering / genetics
SUMO-1 Protein / genetics
SUMO-1 Protein / metabolism
Small Ubiquitin-Related Modifier Proteins / genetics
Small Ubiquitin-Related Modifier Proteins / metabolism
Sumoylation / genetics*
Ubiquitin-Conjugating Enzymes / genetics
Ubiquitin-Conjugating Enzymes / metabolism
Ubiquitin-Protein Ligases / genetics
Ubiquitin-Protein Ligases / metabolism

Substances

AGO2 protein, human
Argonaute Proteins
Molecular Chaperones
Nuclear Pore Complex Proteins
RNA, Small Interfering
SUMO-1 Protein
Small Ubiquitin-Related Modifier Proteins
ran-binding protein 2
Ubiquitin-Conjugating Enzymes
Ubiquitin-Protein Ligases
Lysine

Grants and funding

This work was supported by grants from Ligue Nationale Contre le Cancer (LNCC, Equipe Labellisée), Odyssey-RE, Institut National du Cancer, L’Agence Nationale de la Recherche and European Research Council. P.L. was supported by the LNCC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.