On the other hand, it is tempting to speculate that binding of Dicer to Ago2 may sterically affect accessibility of Lys402 to sumoylation enzymes, in turn influencing Ago2 stability. Tyr393, which is phosphorylated in hypoxia, is also located in the L2g1 linker region, and when phosphorylated, reduces Dicer binding. Phosphorylation at Tyr393 could impact on Lys402 spatial position or accessibility. In line with this hypothesis, we have observed significant reduction in the extent of Ago2 sumoylation when Tyr393 was mutated. Future studies will be necessary to assess biochemical consequences of this potential cross-regulation between Tyr393 phosphorylation and Lys402 sumoylation, as well as its structural and mechanistic basis. Although we have not detected an effect of Lys402 mutation or of impaired cellular sumoylation on siRNA- or miRNA-guided RISC activity as determined by GFP reporter assays, the possibility remains that other aspects of RNA-mediated gene silencing, as analysed in different settings, may be affected by sumoylation. Indeed, Ago2 which has a preference for siRNAinitiated endonucleolytic cleavage, mediated almost complete silencing of the siRNA-based let-7-GFP reporter. This makes it difficult to assess potential effects of ‘more stable’ Ago2 on let-7-GFP which, in theory, is expected to further enhance cellular siRNA function. Yet, the latter is already baseline maximal in our settings. Future studies should determine whether Lys402 sumoylation could impact on Ago2 three-dimensional architecture or on the ability of Ago2 to recruit proteins forming the RISC complex, in turn affecting siRNA or miRNA loading to Ago2 and/or leading to altered siRNA- or miRNA-guided RISC function. Despite our efforts, we were unable to detect a substantial amount of endogeneous SUMO-modified Ago2 in untreated cells or cells undergoing stress such as c-irradiation or arsenic treatment. This implies that sumoylation of Ago2 may occur either transiently in response to a yet unidentified signal/ stress, and/or affects only a very small fraction of Ago2. Indeed, detecting endogenous sumoylation, especially by SUMO1, is technically challenging. In vivo, unconjugated SUMO1 is limiting and exists almost entirely conjugated to high-affinity targets such as RanGAP1, implying that endogenous de novo sumoylation, particularly by SUMO1, involves molecular competition. In most cases only a tiny fraction of target proteins is subjected to sumoylation. The sumoylated fraction may be limited to a specific sub-cellular compartment or to a specific cell type/tissue. Finally, sumoylation is a highly dynamic and transient process representing a constant competition between SUMO-conjugating and deconjugating enzymes. In line with these arguments, sumoylation of HP1a has recently been shown to be highly transient and SUMOmodified HP1a was found to be exclusively enriched at pericentric heterochromatin, highlighting the difficulty of detecting endogenously sumoylated fraction in the whole pool of a given substrate. The evolutionary conservation of the SUMO-acceptor lysines on Ago2 proteins across various species, as well as between Ago1 and Ago2 proteins, point to the importance of Ago2 sumoylation in endogenous settings. In this context, it will be interesting to determine the fraction of endogenous Ago2 modified by SUMO in different sub-cellular locations and protein complexes, in response to stress or pathological conditions. Several lines of evidence suggest that the SUMO E3 ligase RanBP2 may be 6β-Hydroxytestosterone important in fine-tuning Ago2 sumoylation and stability in vivo. RanBP2 enhances Ago2 sumoylation in vitro, colocalizes with Ago2 in the nucleus and antagonizes Ago2 steady-state protein level. Importantly, we have not observed a significant effect of PIAS family of E3 ligases on Ago2 sumoylation suggesting that Ago2 SUMO conjugation may specifically be favored by RanBP2 in vivo. Ubc9 is upregulated in an increasing number of malignancies such as melanoma, breast cancer and ovarian carcinoma, representing a potential target for cancer therapy. In line with our data, a recent report demonstrated that melanoma cells display strong reduction of Ago2 expression on the protein level, while the mRNA level is unchanged, implying post-translational control. Future studies should evaluate contribution of hypersumoylation to Ago2 protein reduction in melanoma and in other malignancies, which likely contributes to deregulation of miRNA expression in cancers. The interconnection of post-translational modifications has emerged as an essential mechanism that governs the spatiotemporal regulation of signaling pathways. Moreover, their reversible nature constitutes a versatile mechanism to adapt to most challenges faced by eukaryotic cells. Our results, which identify sumoylation as a novel post-translational modification acting on Ago2 add another layer of complexity to posttranslational regulation of the RNA interference machinery. This may be the first sign of a complex RNA interference-SUMO interactome. Future studies should determine whether other enzymes of the siRNA/miRNA pathway are subject to sumoylation, and also reveal potential cross-talks between these modifications to regulate RNA interference. 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