Genetic and molecular analysis of germ granule components in C. elegans

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DESCRIPTION (provided by applicant): Germ cells are of clear importance for the maintenance of species and differ from differentiated somatic cells in several fundamental ways. Despite their significance as a cell lineage, precisely how they acquire and maintain their unique characteristics is unknown. Germ granules are a conserved and unique component of germ cells in vertebrates and invertebrates. Several germ granule components are required for fertility; however, their biochemical functions are unknown. This proposal addresses the hypothesis that germ granule proteins interact with mRNA to regulate its translation and/or stability. Molecular, genetic, and cell biology techniques will be used in the nematode, C. elegans, to address two specific aims. First, the significance of RNA localization to germ granules will be determined, using pos-1 mRNA, a previously identified germ granule RNA. In other species, specific 3' UTR regions of mRNAs are known to be necessary and sufficient to target RNA to germ granules. In C. elegans two conserved elements in the pos-1 3'UTR have been identified; site-directed mutagenesis will be used to generate mutations in these regions. Transgenic lines will be made using a GFP reporter fused to various forms of the pos-1 3' UTR. Effects of mutations in the 3' UTR on RNA localization to germ granules, RNA stability, and translational regulation will be assayed to determine how 3' UTR elements and RNA localization affect RNA stability and translational regulation. The second specific aim addresses the function of germ granules in oocytes when oogenesis is arrested. Arrested oogenesis occurs in old-aged wild-type worms and in several mutants that lack functional sperm. Giant aggregates of RNA and germ granule proteins reversibly form in arrested oocytes. Upon fertilization, the aggregates rapidly dissociate. Importantly, the arrested oocytes with aggregates are viable if fertilized and thus, the hypothesis is that aggregates function positively to modulate RNA stability or translational regulation. Experiments will determine the cues that regulate the formation and dissociation of aggregates and identify and characterize mutants defective in aggregate formation. A better understanding of how C. elegans germ cells function may be applicable to germ cells in other organisms as well as to stem cells, which share several characteristics with germ cells. These studies may also uncover conserved mechanisms that oocytes use to preserve their integrity over time and are critical for fertility.