Project title: Molecular mechanism of piRNA biogenesis Project summary Non-coding RNAs have diverse functions in eukaryotic cells. Use of these non-coding RNAs in therapeutic approaches is a promising but rather unexplored direction in biomedical research. We discovered a new class of small non-coding RNAs, piwi-interacting RNAs (piRNAs), that together with their protein partners, Piwi proteins, recognize and silence endogenous genomic parasites called transposable elements and are involved in regulation of host gene expression. The silencing of transposons is critical in germline cells and the failure of piRNA-mediated repression leads to sterility in both Drosophila and mice. The mechanism of biogenesis of piRNAs appears to be distinct from that of other classes of small non-coding RNAs, microRNA and siRNA. piRNAs are encoded in distinct genomic regions dubbed piRNA clusters that produce long ncRNA transcripts, pre-piRNAs, that are further processed to mature piRNAs, which work as guides to recognize and repress RNA targets. In germ cells of Drosophila, dual-strand piRNA clusters are bound by the Rhi-Del-Cuff (RDC) chromatin complex, which is essential for non-canonical transcription of piRNA precursors and acts as a master regulator of piRNA cluster identity. However, how RDC is recruited to the genome to specify regions for piRNA production remains unknown. Our results suggest that piRNAs that are deposited to the egg by the mother guide recruitment of RDC to mark piRNA-generating loci during embryogenesis, and this mark is maintained during later development. After export from the nucleus, piRNA precursors are further processed and loaded into piwi proteins in a cytoplasmic membraneless organelle called nuage. We identified the scaffold protein of nuage and found that a posttranslational modification, symmetric methylation of arginine, of the cytoplasmic piwi proteins plays an important role in both piRNA biogenesis and nuage assembly. We will capitalize on our findings to understand critical steps of piRNA biogenesis in the nucleus and the cytoplasm. We will attack the problem of cluster specification by studying de novo establishment of piRNA clusters and molecular mechanisms of RDC recruitment and maintenance, and study nuage formation and the role that this compartmentalization plays in piRNA biogenesis. Our studies will help to advance our understanding of the mechanism of transposon silencing, which is important for both fertility and for genomic stability. It will also provide the basis for future use of the piRNA pathway as a tool in research and therapy. Importantly, the significance of the proposed research extends well beyond answering important questions in the non-coding RNA field. Our studies will provide clues to the problems of specification of distinct chromatin domains, decoding of the histone code and formation and function of membraneless cellular compartments. As such, our work will explore fundamental mechanisms that c...