Translational control plays a key role in spatial and temporal regulation of gene expression during development. The proposed research aims to elucidate translational control mechanisms underlying developmental processes, using the Drosophila nanos (nos) gene as a model. nos is ideal for these studies because it encodes a translational repressor whose own synthesis must be highly regulated for proper embryonic development. During late oogenesis and early embryogenesis, patterning of the anterior-posterior body axis requires that nos translation within the bulk cytoplasm be repressed. Translational repression of nos is initiated during oogenesis, mediated by the interaction of the hnRNP F/H homolog Glorund (Glo) with a structured translational control element (TCE) in the nos 3' untranslated region (3'UTR). Glo, like many other RNA-binding proteins, recognizes multiple target RNAs and exerts multiple functions. Since the precise roles of RNA-binding proteins in regulating gene expression are likely to be determined by their RNA binding specificity, it is important to have a clear understanding of how these proteins interact with their RNA targets as well as how these interactions transduce a particular biological response.
Aim 1 combines functional genetic analysis and structural biology to validate and extend the model for Glo?TCE interaction developed during the current grant period. Unbiased identification of Glo interacting RNAs and proteins will decipher how Glo?RNA interactions confer regulation and will elucidate the relationship between RNA recognition, regulatory activity, and biological role.
Aim 2 uses in vitro translation and ribosome footprinting assays to probe the molecular mechanisms by which the TCE?Glo interaction inhibits translation, and examines the broader translational landscape to uncover previously unappreciated developmental regulation.
Aim 3 is motivated by results from the current funding period that highlight the importance of post-transcriptional control in dendrite morphogenesis. Results from neuronal knockdown of RNA-binding proteins in an RNAi screen set the stage for determining the specific pathways controlled by these proteins and, ultimately, the RNA targets whose regulation is essential to control dendrite growth and branching. In addition, a model for local protein synthesis in dendrites will be tested by direct visualization of nos translation in vivo. Mutations in translational regulatory proteins have been associated with a variety of cancers and diseases with neurological dysfunction. The proposed work will provide fundamental insight into how these factors control development, growth and differentiation, and how the disruption of translational control can result in disease.
Control of mRNA translation enables cells to rapidly start or stop making proteins and to restrict where in the cell particular proteins are produced. This flexibility is particularly important in animal development, from the formation of eggs to the differentiation, growth, and function of specialized cells like neurons. Mutations in proteins that control mRNA translation have been associated with a variety of cancers and diseases with neurological dysfunction; thus, by investigating how translation is controlled and the factors that control it, the proposed work will provide fundamental insight into the deployment of translational control for development, growth and differentiation, and how its disruption can result in disease.
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