PROJECT SUMMARY/ABSTRACT Reproductive success of any species relies critically on germ cell development. Germ cells are totipotent cells that transfer their genome form one generation into the next. Upon fertilization, they differentiate into all somatic lineages while setting aside a fraction of cells that give rise to future germ cells. They realize this feat by establishing a germline specific genomic landscape through transcriptional repression and chromatin remodeling coupled with an expansive post-transcriptional regulation involving mRNA localization. Germ cells form germ granules and all organisms depend on these granules for germ cell function. mRNAs enrich within granules where their translation and degradation is regulated. First, critical steps of germ cell development rely entirely on post-transcriptional events such as mRNA localization. This process is fundamental in achieving spatial and temporal control of mRNA translation and stability not only during Drosophila germ cell specification but also during mammalian spermatogenesis. Thus, to understand germ cell development and the reproductive success of any species it is paramount to characterize the mechanisms that govern mRNA localization to germ granules. Lack of adequate spatial and temporal sensitivity to resolve single mRNAs specifically in the germline in intact organism through development has profoundly hindered our ability to fully understand the mechanisms and the impact of mRNA localization on germ cell development. Using a genetically amenable model organism such as the fruit fly, coupled with biochemistry and single-mRNA imaging, provides an ideal strategy to complement mammalian research. During my postdoctoral studies, I developed a super-resolution approach where I coupled single-mRNA fluorescent in situ hybridization (smFISH) with structural illumination microscopy (SIM) and showed that multiple mRNAs form homotypic clusters that are asymmetrically- distributed in granules. In contrast, proteins were evenly distributed within granules. My hypothesis is that the mRNA itself plays an instructive role in mRNA localization and germ granule assembly. Here, I present a focused strategy to address my hypothesis and characterize the formation of homotypic clusters and germ granules in vitro and in vivo. With much improved resolution over SIM, I will implement stochastic optical reconstruction microscopy (STORM) super-resolution and total internal reflection fluorescence (TIRF) microscopy to dissect how mRNA clusters form in the embryo and in reconstructed germ granules in Drosophila S2 cells, identify sequences that drive clustering and identify the biological relevance of clustering. I will then identify proteins that promote higher-order RNA clustering. Finally I will examine how mRNAs and granule proteins promote granule assembly. The knowledge gained in this study will provide understanding of how mRNA localization shapes germ cell development. Given that many features of germ cell development are conserved among species, any genes, networks and mechanisms identified as well as technologies developed could be implemented to study germ cell development and reproduction in other organisms.
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NIH ExPorter