Cells respond to extracellular stress by inducing specific intracellular programs through the activation of stress kinase signalling pathways. Stress responses are associated with the phosphorylation of particular substrates implicated in restoring homeostasis and cell integrity. Oncogene activation has also been shown to generate replicative DNA damage, activate the DNA Damage Response (DDR) and thereby limit the expansion of precancerous cells. At the molecular level, the DDR is a functional network of different proteins that cooperate in arresting the cell cycle and repairing DNA damage, or inducing cell death if the damage exceeds repair capacities. Recently, micro (mi)RNAs were shown to regulate the expression of several DDR proteins. However, it is not known whether the activity of miRNAs is affected by the stress response. miRNAs are a class of small regulatory RNAs that repress translation and induce degradation of their target mRNAs. Argonaute (Ago) proteins are essential for miRNA activity by forming the central component of the RNA-induced silencing complex. Preliminary data from the Stoecklin lab show that Ago-2 and miRNA-dependent mRNA decay are inhibited in response to oxidative stress, one of the major causes for DNA damage. We propose to investigate the mechanisms of Ago-2 regulation in response to oxidative stress and other DNA damage agents, with the aim to identify possible phosphorylation sites in Ago-2, elucidate the signalling pathways involved, and examine the physiological consequences of Ago-2 inhibition on the cell cycle, the apoptotic response and cell transformation. We will further address specific interactions that Ago-2 undergoes under conditions of oxidative stress with the phosphatase PP6c, the proteasome and the SMN complex. Taken together, the proposed work will elucidate mechanisms that control Ago-2 function and miRNA activity, which will have significant impact on our understanding of RNA interference.