Intracellular mRNA localization is an important mechanism for the spatial regulation of gene expression necessary for animal development. By restricting the distributions of developmental regulatory proteins to particular regions of oocytes and embryos, mRNA localization plays a critical role in patterning of body axes and specification of cell fates during embryonic development in a variety of organisms. In addition, RNA localization generates protein asymmetries necessary for the subsequent differentiation and function of many specialized cell types. Cis-acting signals that mediate localization have now been identified in a number of localized mRNAs. Little is known, however, about the mechanisms by which these signals are recognized specifically by cellular localization machinery and how they target their RNAs to unique intracellular locations. ? ? Restriction of Nanos protein to the posterior of the Drosophila embryo is essential for proper patterning of the anterior-posterior body axis. Nanos synthesis is limited to the posterior pole of the embryo by a combination of RNA localization and translational control. Localization of nanos mRNA to the posterior pole of the embryo generates the critical concentration Nanos protein in the posterior for abdominal development and is essential to activate nanos translation. ? ? Localization of nanos is mediated by a complex a cis-acting localization signal within its 3' untranslated region (3'UTR). Results from previous and preliminary studies indicate that cytoplasmic localization factors recognize different sequence or structural motifs within this localization signal. Using nanos as a model, the proposed work will provide insight into how complex RNA localization signals are recognized by the cellular localization machinery and how these RNA-protein interactions mediate transport and anchoring by localization pathways. More generally, these studies will shed light on mechanisms by which RNA-protein interactions provide the highly selective control of basic cellular processes needed for development, growth, and differentiation.
Specific Aim 1 encompasses mutational analysis of the nanos localization signal to determine sequence and structural requirements for localization signal recognition and function. This work will be facilitated by phylogenetic analysis ofnanos 3'UTRs from ten different drosophilid species. Preliminary work has led to purification of one candidate nanos localization factor and biochemical identification of a second.
Aim 2 focuses on biochemical and genetic characterization of these factors to determine their function in nanos localization. In addition, a new strategy for biochemical isolation of localization complexes is proposed.
In Aim 3, a genetic screen for nanos localization factors will complement the biochemical approaches of Aim 2.
Aim 4 takes advantage of a new system for GFP labeling of nanos RNA in vivo to investigate the dynamic pathway of nanos localization during oogenesis. ? ?
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