Determining the role of a TENT5 family non-canonical polyA polymerase in Drosophila spermatogenesis - PROJECT SUMMARY/ABSTRACT Translational repression and reactivation play critical roles in the development and homeostasis of multicellular organisms. These processes have been best studied in oocytes, which are often generated but not fertilized for weeks, months or even years. Studies of Xenopus oocytes have shown that translational repression and reactivation of at least some mRNAs involves removal of the mRNA polyA tail and coupling with proteins for storage, followed by regeneration of the polyA tail for translational reactivation after egg activation by progesterone. The TENT2 family of non-canonical polyA polymerases has been linked to this process in Xenopus as well as in other animals. In Drosophila melanogaster, mRNAs must also be translationally repressed and reactivated during spermatogenesis. Our preliminary data shows that a member of a different family of non-canonical polyA polymerases, TENT5 is required for spermatogenesis in Drosophila melanogaster. TENT5 family members have been linked to spermatogenesis in mice and humans as well, but the mechanisms are unknown. In D. melanogaster, as in vertebrates, spermatogenesis occurs in a syncytium through meiosis and most of spermatid differentiation. We have generated a strong loss-of-function CRISPR- Cas9 allele, TENT52-83 that affects the end of spermatogenesis, after meiosis, when the spermatids become separated into individual cells, a process termed spermatid individualization. In this proposal, we describe our plans to define the role of D. melanogaster TENT5 in spermatid individualization. In Aim 1, we will use in situ hybridizations to determine the TENT5 expression pattern in the testes and use Gal4/UAS-based approaches to knock down TENT5 in the germline or the soma. This approach will allow us to determine in which cell type TENT5 is required. In addition, we will attempt to rescue the TENT52-83 phenotype with wild type and catalytic- dead versions of TENT5, to determine if TENT5 requires its polyA polymerase catalytic domain for its role. In Aim 2, we will examine the localization of proteins with known roles in spermatid individualization, whose mRNAs might be targets of TENT5 polyadenylation. We will use established techniques to pinpoint the stage at which TENT52-83 affects spermatid individualization. The results of these analyses are expected to lay the groundwork for future studies to confirm TENT5 targets with single molecule fluorescent in situ hybridizations and co-staining with TENT5 protein, as well as RNA immunoprecipitation, for instance, that will define the mechanism by which TENT5 proteins function in spermatogenesis, including potentially in humans.
