Among couples trying to conceive, 10-15% will be affected by infertility, which can be caused by impaired gametogenesis. Effective post-transcriptional gene regulation by RNA-binding proteins (RBPs) is crucial for normal gametogenesis, with mutations in RBPs impairing such regulation and causing infertility in humans. For example, the Deleted in Azoospermia (DAZ) family of RBPs are essential for human fertility, because during spermatogenesis they regulate translation of a subset of mRNAs by binding to their 3′ UTRs; their molecular mechanism of action was understood only after their Drosophila ortholog was identified and studied genetically. However, the full repertoire of RBPs that are essential for gametogenesis is currently unknown. To fully understand the complex post-transcriptional gene regulation during gametogenesis, it is critical to identify novel RBPs that are important for these processes and elucidate their molecular functions and mechanisms of action. We have recently identified three previously uncharacterized RBPs (MARF1, Tanenashi, and CG44249) that are essential for fertility in Drosophila. Our data suggest that MARF1, which is expressed in oocytes and is required for female fertility, regulates mRNA poly-A tail length during oogenesis. Tanenashi, which is expressed in spermatocytes and is required for male fertility, plays roles in promoting the expression and splicing of male fertility factor genes containing mega-base-sized introns with highly repetitive DNA sequence on the Y chromosome during spermatogenesis. CG44249, which is expressed in ovaries and is required for female fertility, likely plays a role in splicing regulation during oogenesis. We also showed that a fourth RBP (Loqs-PB), which is important for female fertility and germline stem cell maintenance in ovaries, regulates the length of microRNAs in ovaries. These four RBPs are conserved from flies to humans. Using Drosophila, I now propose to further investigate these four RBPs, and determine their precise molecular functions and mechanisms of action in gametogenesis. By investigating mutant flies in vivo and the proteins in vitro, we will test our hypothesis that: (i) these RBPs each bind specific target RNAs by recognizing specific RNA sequence and/or structure motifs, and (ii) thereby promote or inhibit the processing of bound RNAs at specific molecular steps, allowing highly regulated spatiotemporal expression of targeted genes during gametogenesis. These studies will advance our fundamental knowledge of the molecular mechanisms of post- transcriptional regulation of gene expression, especially by regulating the mRNA poly-A tail length, mRNA splicing, and microRNA length, during gametogenesis, providing potential for improved diagnosis and treatment of human infertility.