Plants and animals use small (s)RNA to control many developmental and physiological processes. sRNAs associate with ARGONAUTE (AGO) proteins as part of RNA-induced-silencing-complexes (RISC) to regulate gene expression at the transcriptional or post-transcriptional levels. The major effector of post-transcriptional gene silencing (PTGS) in Arabidopsis thaliana, AGO1, has been mainly implicated in cytoplasmic processes including cleavage (or ’slicing’) and translational repression of sRNA target transcripts, as well as silencing of cytoplasmically replicating viral RNA. However, several lines of recent evidence, including from the host laboratory, suggest the existence of as yet unidentified functions for AGO1 in the nucleus; conclusive evidence for nuclear PTGS was, moreover, recently obtained in soybean. This proposal outlines a series of mutually reinforcing experiments aimed at deciphering putative nuclear functions of Arabidopsis AGO1. By combining biochemical fractionation methods, AGO1 nuclear interactome exploration, deep-sequencing analyses, cell biology and forward genetics, the proposed project will: 1) analyze the nature of sRNA bound to nuclear AGO1 and identify their origin and targets; 2) study the possible contribution of nuclear AGO1 to antiviral defence and transcriptional gene silencing, and 3) explore the possible occurrence and extent of nuclear slicing. The results are expected to profoundly modify our current views of RNA silencing in plants, and perhaps in metazoans. Implications in agriculture are also anticipated, as the project may elucidate novel aspects of antiviral defence and plant developmental patterning. Finally, the knowledge on cell fractionation procedures, large-scale RNA analyses and Arabidopsis AGO biology generated during the project will contribute to European excellence and competitiveness in the field.