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.


Gene expression analyses
Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen) according to the standard procedures. For Ubc9 and Ago2 transcript level analysis, total RNA was reverse transcribed using Applied Biosystems high capacity cDNA kit. Ago2 and Ubc9 expression levels were normalized to EF1/Ezrin expression.

Determination of Ago2 stability
For measurement of Ago2 half-life, cells were treated for up to 24 hrs with cycloheximide (CHX) at a concentration of 50 µg/mL. Cells were harvested at indicated times after treatment, lysed in Laemmli buffer and analysed by SDS-PAGE. The intensity of each band was evaluated by densitometry using the Image J software and Ago2 expression level was normalized to tubulin expression.

Nucleo-cytoplasmic fractionation
PBS-washed cell pellets were resuspended in 10 volumes of buffer 1 (0.5 M sucrose, 15 mM Tris [pH 7.5], 60 mM KCl, 0.25 mM EDTA, 0.125 mM EGTA, Protease inhibitor cocktail tablet [Roche]). The cells were then allowed to recover for 5 min at 4°C, 10% NP-40 was added dropwise. The reaction was stopped by adding NP-40 when the nuclei were free of cytoplasmic contaminants. The lysed cells were centrifuged at 1,000 g. The supernatant (cytoplasmic fraction) was decanted, and the pellet (corresponding to the nuclei) was resuspended in extraction buffer (20 mM Tris-Cl [pH8.5], 10% glycerol, 2 mM dithiothreitol, 0.8 M KCl). The DNA was sheared by passage through a 25-gauge syringe and incubated for 45 min on ice. The mixture was centrifuged at 21,000 g, and the nuclear fraction was dialyzed against 150 mM KCl in extraction buffer. The various cellular fractions were analyzed by Immunoblotting.

Immunofluorescence
HeLa cells or MEFs were seeded on coverslips. Cells were fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature and subsequently permeabilized by 0.1% Triton X-100. The samples were then incubated overnight at 4°C with indicated primary antibodies.

RNA interference
Ubc9, RanBP2 and SAE2 expression was ablated by an siRNA pool (Dharmacon, On-target Plus). siRNAs (50 nM final concentration) were introduced into HeLa cells using Lullaby transfection reagent (OZ Biosciences) according to the manufacturer's instructions. Cells were analysed after 72h. Human HT1080 cells were transfected with a control shRNA (scr) or a shRNA against Ubc9 and selected by puromycin as described previously [22].

Immunoblotting and Immunoprecipitations
Adult mice organs were dissected and homogenized in 1ml of lysis buffer (50mM Tris-HCl Immunoreactivity was visualized using an infrared imaging system (Odyssey; LI-COR Biosciences). Protein quantification was performed using Odyssey and ImageJ (National Institutes of Health) software.
For co-immunoprecipitation of Ago2 and Ubc9, cells were scraped in PBS and lysed in immunoprecipitation (IP) buffer (50 mM Tris, pH 8.0, 0.5% NP-40, 200 mM NaCl, 0.1 mM EDTA, 10% glycerol, and protease inhibitors (Complete EDTA-free, Roche)) supplemented with 10mM N-ethylmaleimide (NEM). Lysates were subsequently incubated for 2h at 4°C with anti-HA antibody and immune complexes were collected by incubation for 2h at 4°C with Protein A/G agarose beads (Calbiochem) and washed three times in lP buffer. The agarose beads were boiled for 5min in 2X Laemmli sample buffer and analysed by SDS-

Endogenous miRNA-guided si/miRISC activity assay
The assay was performed as described in [18]. pcDNA3-GFP, pcDNA3-GFP-let-7 (PE) and pcDNA3-GFP-miR-21 (8×BU) were a kind gift from Y. Shi. Briefly, GFPL and GFP-let-7 or GFP-miR21 expression plasmids were transfected in HeLa cells that were previously transfected with siRNA (scr or against Ubc9) or HA-Ago2 (wild type or mutants) expression plasmids. Cell extracts were analysed by Western blot for GFP expression. For each condition, silencing efficiency was calculated as the percent decrease in GFP expression (from let-7-or miR-21-regulated fusions) compared to unregulated long GFP (GFPL) and control GFP (ctl).