Probing Translational Control during Drosophila Oogenesis: from nanos to the Entire Maternal Transcriptome

Abstract

Translational control provides a key mechanism for the spatial and temporal regulation of eukaryotic gene expression. It plays a particularly important role in early embryonic development in organisms that rely heavily on maternally supplied mRNAs. The Drosophila developing oocyte serves as a valuable model for studying translational control. The Drosophila posterior determinant, nanos (nos), is translationally repressed throughout the oocyte cytoplasm except at the posterior pole. A multi-functional RNA-binding protein Glorund (Glo), the homolog of the mammalian hnRNP F/H family of proteins, represses translation of nos during oogenesis by targeting both translation initiation and translation elongation. To elucidate the molecular mechanism by which Glo regulates nos, I identified dFMRP as a Glo-interacting protein. By biochemically dissecting repression of nos translation in vitro, I demonstrated that dFMRP specifically inhibits translation elongation. Furthermore, I combined mutational analysis and in vivo and in vitro binding assays to show that Glo’s qRRM2 domain specifically and directly interacts with dFMRP, suggesting that Glo’s RNA-binding domains can also function as protein-protein interaction interfaces critical for its regulatory functions. Additionally, I applied ribosome footprint profiling to the Drosophila ovary at different developmental stages to identify maternal transcripts regulated during the translation elongation phase. A footprint peak-finding tool has been developed to detect transcriptome-wide ribosome stalling sites. My preliminary results suggest that developmentally regulated ribosome stalling sites may be widely present on Drosophila maternal transcripts. An A-/P-site mapping algorithm is currently under development to facilitate mechanistic analysis of ribosome stalling during oogenesis.