Subcellular RNA localization is one strategy whereby cells create localized concentrations of proteins required for specific cell structures or overall cell polarity. The long term goal of this research is to understand how cells subcellularly localize their RNA. mRNA for the Drosophila bicoid (bcd) gene is localized at the anterior end of the developing oocyte. Translation of this localized RNA during early oogenesis leads to a concentration gradient of the bcd protein, which is a transcription factor which acts to set up anterior-posterior polarity in the developing embryo. The exuperantia (exu) gene is required for localizing the bcd RNA at the anterior end of the oocyte. We propose to determine how the exu gene product, and also other gene products, function in the localization process. We have preliminary results which show that bacterially-expressed exu protein binds to bcd RNA in vitro, and that binding may be dependent on a small arginine-rich domain in exu; arg-rich domains have been shown to necessary for the binding of other proteins to RNA, such as the HIV Tat protein. Experiments are proposed in this grant to determine the specificity of this binding, and to determine the contributions of the arg-rich domain within exu for RNA binding, and the target sequences within the bcd RNA. A genetic screen will be done to identify other factors which act in bcd RNA localization, and immunoprecipitation experiments will determine if the products of other genes already known to affect bcd RNA localization interact directly with exu protein during oogenesis. Unlike other genes that have been shown to act in bcd RNA localization, exu is also required during spermatogenesis, and may have a function in some somatic cells. Thus, exu may have a more general function, possibly to localize RNAs in these other tissues. In spermatogenesis, RNA targets will be sought, and ectopic expression of bcd in the testis will determine if exu protein there is sufficient for bcd RNA localization. exu produces sex-specifically processed RNAs in the germline, which encode the same predicted protein, but differ in untranslated regions. Our preliminary results suggest that the male-specific 868 3'UTR is required for normal function during spermatogenesis; the function of this region will be analyzed. Finally, exu's function in somatic cells will be analyzed by characterizing the behavioral defects associated with some exu alleles, sequence analysis of an exu mRNA which is specific to somatic cells, and determination of its developmental expression pattern.
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