Identifying RNA-binding Proteins Required for mRNA localization to Drosophila Germ Granules

Germ cells are specialized cells in metazoans that transfer genetic material from parents to the offspring, thus allowing the continuation of the species. In Drosophila, a specialized cytoplasm called the germ plasm accumulate at the posterior pole of the developing embryo, instructing the formation of germ cells. A single protein called Oskar is necessary and sufficient for the nucleation of germ plasm. Contained within the germ plasm are non-membrane bound RNA granules called germ granules. Germ granules enrich approximately 200 different maternally-provided mRNAs that code for proteins required for germ cell development. These mRNAs only translate in granules and are repressed elsewhere in the embryo. It is unclear how most of these mRNAs enrich in germ granules and how they promote germ cell development. Biochemical analysis identified Oskar as an RNA binding protein (RBP) that binds at least three critical germ granule mRNAs, nanos (nos), germ-cell-less (gcl) and polar granule component (pgc), suggesting that Oskar could be recruiting these mRNAs to granules. However, this model has never been experimentally tested. In addition. it is unclear how other germ granule mRNAs enrich in germ granules. The aim of my project is to identify RBPs required for mRNA enrichment to Drosophila germ granules. Immunoprecipitation of Oskar from early Drosophila embryos revealed 47 unique RNA-binding proteins (RBP) as germ granule constituents and possible candidates that could recruit mRNAs to germ granules. To test this idea, an RNA interference (RNAi) screen was performed to assess the role of germ granule RBPs in mRNA recruitment to Drosophila germ granules. Experiments revealed that out of these 47 RBPs, only 4 (Vasa, CG7878, HnRNP-K and Kra) appear required for mRNA recruitment. The rest participate in the maintenance of germ granules (4) and germ granule assembly and disassembly (14). Moving forward, I will test whether Vasa, CG7878, HnRNP-K and Kra are also sufficient to recruit germ granule mRNAs to an ectopic location in the embryo to further validate the involvement of these proteins in mRNA enrichment to germ granules. My work will help elucidate the molecular regulators and mechanism of mRNA enrichment to Drosophila germ granules