PROJECT SUMMARY Transposable elements (TEs) are “genomic parasites” that can replicate and re-integrate into the host cell genome. Uncontrolled TE activity in germ cells leads to DNA damage, disruption of gametogenesis, and infertility. In mammalian male germ cells, the PIWI- piRNA pathway uses small RNAs as a guide to silence mobile TEs to protect genome integrity and sustain fertility. The proper production of piRNAs is critical for TE silencing and spermatogenesis. However, the mechanisms governing piRNA biogenesis are not well understood. In particular, the roles of many RNA binding proteins during piRNA biogenesis remain elusive. By studying a subgroup of Tudor domain proteins that also harbor LOTUS domains, we discovered that TDRD5 is a novel RNA binding protein critical for piRNA biogenesis in mice. Strikingly, we have discovered a novel RNA binding property of LOTUS domains that is conserved in bacteria, plants and animals. This binding property is different from reported protein binding feature of some animal LOTUS domains. We hypothesize that animal LOTUS domains have both RNA and protein binding activities and that the LOTUS group of Tudor domain proteins together play critical roles in mammalian piRNA biogenesis. To test this hypothesis, we will use biochemical approaches and mouse models to: 1) Clarify the RNA and protein binding activities of the LOTUS domain superfamily; 2) Determine the mechanism of a specific LOTUS domain-RNA interaction; and 3) Define the functional involvement of LOTUS domain proteins in piRNA biogenesis and Vasa regulation in mice. These studies will provide valuable new insights into the mechanisms of piRNA biogenesis that safeguard germline genome integrity to sustain male fertility and expand our knowledge in broader RNA biology and developmental biology.