Transposons are parasitic DNA elements that when activated can insert into new genomic locations, leading to genomeinstability. Animal germlines express a special class of small RNAs called piwi-interacting RNAs (piRNAs) which areimplicated in transposon silencing. In mammals, they are believed to promote DNA methylation of transposon-rich genomicregions. Mechanisms of piRNA biogenesis and function are only beginning to be understood. Sequence analysis of piRNAsfrom a variety of organisms has given rise to working models for piRNA biogenesis and how piRNAs might act as guidesfor nuclear silencing complexes. However, key components of the pathway, especially those for biogenesis and function,remain to be discovered. Our aim is to identify and characterize new components using a combination of biochemistry, mousegenetics and small RNA bioinformatics. The regulatory potential of posttranslational modification of piwi proteins and theirrecognition by tudor proteins will be examined. Mouse mutants will be used to study the in vivo role of catalytic activity ofpiwi proteins in piRNA biogenesis and identify putative targets by transcriptomics approaches. Integrated biochemical anddeep-sequencing methods will be applied to understand how small RNAs might guide nulcear silencing machinery to targetlocations. Finally, in a field that is dependent on model organisms, we propose to develop a cell culture system to study thepiRNA pathway and carry out a high-throughput functional RNAi screen for component discovery. This proposal aims touse interdisciplinary approaches in uncovering the biochemical framework in which germline small RNAs function to protecteukaryotic genomes.