Proust traverse le temps

Marcel Proust en 1900 © DR Une explication littéraire est-elle indispensable pour percevoir le degré de composition de l’œuvre ? A.C. À la parution, beaucoup de lecteurs ont dit : « C’est écrit au fil de la plume, ce sont des mémoires, du bavardage… ». Un bon lecteur comme Jacques Rivière, secrétaire général de La Nouvelle Revue française (NRF), a compris, lui, qu’au contraire ce livre était extrêmement composé. « Rien n’est là par hasard », disait Proust : si telle chose a lieu à tel moment,..

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

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

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation

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Extensive SUMO Modification of Repressive Chromatin Factors Distinguishes Pluripotent from Somatic Cells

International audiencePost-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development

Host-Specific Myrmecophily and Myrmecophagy in the Tropical Coccinellid Diomus thoracicus in French Guiana

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Impaired global sumoylation enhances Ago2 protein levels in cells and in mice

<p>(<b>A</b>) Depletion of SUMO enzymes results in increased steady-state Ago2 protein levels. HeLa cells were transfected with control siRNA (scr) or siRNA against Ubc9, SAE2 or RanBP2. Relative levels of Ago2 are indicated in the graph. Means of three independent experiments. Significant p-values are shown. (<b>B</b>) Loss of Ubc9 <i>in vivo</i> results in enhanced endogenous Ago2 protein levels. <i>Ubc9^+/+/ROSA26-CreERT2</i> control (Ubc9^+/+) and <i>Ubc9^fl/−/ROSA26-CreERT2</i> conditional knockout (Ubc9^−/−) adult mice (n = 3, #1, #2, #3) received 3 consecutive days of 4-OHT injection. Proteins were extracted from heart, liver and skeletal muscle and were analyzed by Western blot. Quantification of Ago2 protein levels in organs after normalization to the ponceau staining is shown. Bars represent means and standard deviations. from 3 mice (white bars: Ubc9^+/+, black bars: Ubc9^−/−). P-values, determined by two-tailed Student’s t-test, are indicated.</p

Mapping of the SUMO-acceptor sites on Ago2.

<p>(<b>A</b>) <i>In silico</i> prediction of sumoylation sites on human Ago2. Inspection of human Ago2 amino acid sequence using SUMOsp software reveals existence of four ΨKxE/D sumoylation consensus motifs. (<b>B</b>) Conservation of the four sumoylation consensus motifs (red square) on Ago2 proteins across different species. Amino acid sequences from Uniprot database were aligned using Clustalw software. (<b>C</b>) Lysine 402 of Ago2 is the major SUMO conjugation site. HeLa cells were transfected with plasmids expressing wild-type Ago2, Ago2-K62R, Ago2-K266R, Ago2-K402R, Ago2-K693R or Ago2-K62R/K266R/K402R/K693R (Ago2-4KR) in the presence of Ubc9 and either SUMO1 (left panel) or SUMO2 (right panel) and followed by Western blotting using an anti-Ago2 antibody. Representative gels from three independent experiments. (<b>D</b>) Quantification of Ago2 sumoylation in the context of wild-type Ago2 or its mutants. Extent of SUMO-conjugated Ago2 was assessed after normalization to total Ago2 levels and loading. Means and standard deviations from three independent experiments, as well as statistically significant decreases in sumoylation are shown.</p

Sumoylation negatively regulates Ago2 turnover.

<p>(<b>A</b>) Sumoylation-deficient Ago2 mutants show increased half-lives. HeLa cells transfected with HA-Ago2-wt, HA-Ago2-4KR or HA-Ago2-K402R were treated with 50 µg/mL of cycloheximide (CHX) for the indicated times. The blot shown is a representative of four independent experiments. The cumulative results of are displayed on the graph (right panel). The initial levels of wild-type Ago2, Ago2-4KR and Ago2-K402R were normalized to 100%. Means and standard deviations are indicated (n = 4). (<b>B</b>) Endogenous Ago2 displays enhanced stability in Ubc^−/− MEFs in which global sumoylation is impaired. The blot shown is a representative of three independent experiments. CHX treatment and quantifications were performed as in A. Western blots for tubulin, Ubc9 and unconjugated SUMO1 are shown as controls. (<b>C</b>) Transient knockdown of Ubc9 results in enhanced Ago2 protein levels. HeLa cells were transfected with either a control siRNA (si-scr) or a siRNA targeting Ubc9 (si-Ubc9). Ago2 relative expression to tubulin is presented in the middle graph and normalized to 100% for siRNA-scr transfected cells. Means of three independent experiments. Graph on the right shows that Ubc9 knockdown has no effect on Ago2 mRNA expression. Total RNA extracted from HeLa cells transfected with si-scr or si-Ubc9 were analyzed by qRT-PCR with primers specific for Ubc9 and Ago2 mRNA. (<b>D</b>) Stable knockdown of Ubc9 results in enhanced Ago2 protein levels. Human HT1080 cells transfected with a control shRNA (scr) or a shRNA against Ubc9 were puromycin selected and processed for immunobloting with anti-Ago2 and anti-SUMO1 antibodies. Ago2 relative expression was quantified. Graph represents means of three independent experiments.</p

<i>In vivo</i> and <i>in vitro</i> sumoylation of human Ago2.

<p>(<b>A</b>) Ago2 is modified by SUMO1 and SUMO2 <i>in vitro</i>. ³⁵S-labelled <i>in vitro-</i>translated Ago2 was incubated in a sumoylation mix containing purified SAE1–SAE2, Ubc9 and SUMO1 or SUMO2, in the absence (-) or presence (+) of ATP. Reaction products were visualized by SDS–PAGE and autoradiography. (<b>B</b>) Sumoylation of Ago2 <i>in vivo</i>. HeLa cells were transfected with expression vectors for HA-tagged Ago2, SUMO1, His-SUMO1, SUMO2 and His-SUMO2 as indicated and analyzed by Western blot by anti-Ago2 antibody. (<b>C</b>) Ubc9 interacts with Ago2 <i>in vivo</i>. HeLa cells were transfected with expression vectors for HA-Ago2 and Ubc9 as indicated. Cell lysates were immunoprecipitated (IP) with mouse anti-HA antibody or control antibody (IgG) and probed with anti-HA and anti-Ubc9 antibodies. WCE, whole-cell extract, 2% of amount used in IP. (<b>D</b>) SUMO E3 ligase RanBP2 enhances sumoylation of Ago2 <i>in vitro</i>. <i>In vitro</i> sumoylation of ³⁵S-labelled, <i>in vitro</i>-translated human Ago2 in the absence or presence of RanBP2 or GST control. Reactions were incubated for indicated times (t = 0 min or t = 60 min) with 10 ng (1x) or 3 ng (0.3x) recombinant Ubc9. Reaction products were visualized by SDS–PAGE and autoradiography. Each blot is a representative of three independent experiments.</p

Subcellular localization of SUMO-conjugated Ago2.

<p>(<b>A</b>) Sumoylated Ago2 resides both in the cytosol and nucleus. HeLa cells were transfected with the indicated plasmids and fractionated to analyze nuclear and cytoplasmic proteins. HA-Ago2 was detected by anti-HA antibody. Immunoblots for endogenous PARP1, a nuclear protein, and endogenous vinculin, a cytoplasmic protein, were performed to validate nuclear-cytoplasmic fractionation. (<b>B</b>) Sumoylation is dispensable for subcellular localization of Ago2. HeLa cells were transfected with wild type or sumoylation mutants (4KR and K402R) of HA-Ago2, and treated as indicated (250 µM sodium arsenite -NaAsO₂- for 60 min). Ago2 localization was determined by immunofluorescence using the anti-HA antibody (red). The cells were also immunostained for Dcp2 (marking P-bodies in green). The nuclei are stained with DAPI (blue). Ago2 staining was diffuse in the cytoplasm, as well as concentrated in cytoplasmic P-bodies (marked by arrows in untreated cells). NaAsO₂ treatment increases size and number of P-bodies. (<b>C</b>) Endogenous Ago2 (red) and RanBP2 (green) colocalize in the nuclei of HeLa cells (indicated by arrowheads).</p