Phosphorylation at Tyr393 could TC 1 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 SMBA 1 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.