RNA localization allows eukaryotic cells to control gene expression in space and time and contributes importantly to cell polarization, allowing asymmetry in events such as cell division, oogenesis and body patterning. Major contributions to our understanding of the functional aspects of RNA regulation control have come from studies in Drosophila. Investigation into oskar mRNA localization in the oocyte, which is essential for germ cell development in the embryo, has revealed a key role of the Exon Junction Complex (EJC) RNA transport. In contrast, in mammals the main role of the EJC shown to date is in RNA quality control, in nonsense-mediated decay (NMD) of mRNAs containing premature stop codons. However, recent reports have linked the EJC to development and cell polarization in mammals as well. The proposed project aims to determine the full spectrum of EJC functions in RNA regulation, using Drosophila as a model. This will be addressed by creating transgenic Drosophila strains expressing tagged-EJC fusions for use in biochemical assays. Purified EJC complexes will be analyzed by SOLEXA based deep-sequencing and SILAC based mass spectrometry following two major objectives: 1) to investigate the EJC associated transcriptome in the oocyte and selected tissues during development and 2) to define the oskar mRNA-associated EJC proteome and its composition at different exon-exon junctions and at different stages of its localization. The results obtained with this unbiased approach will constitute a major step forward in understanding EJC function, not only in the fly, but also in mammals. The project’s multidisciplinary nature, combining cutting-edge biochemical, genomic, and Drosophila genetic techniques, will provide a unique opportunity to generate new scientific knowledge and acquire novel experimental skills that will be essential bases for future research and an independent scientific career.
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