Sub-cellular localisation of RNA has emerged as a key mechanism through which cells become polarized. The localisation of transcripts is an extremely efficient way to target gene products to subcellular compartments or to specific regions of a cell or embryo, making it an important posttranscriptional level of gene regulation. The aim of this project is to perform an RNA-interference (RNAi) screen to identify novel components required for intracellular mRNA transport in Drosophila. A wide range of Drosophila cell-lines will be characterized to select those with asymmetric morphology well suited to visualise localised transcripts. Initially, I will screen for dsRNAs that disrupt RNA localisation as assayed by sensitive fluorescence in situ hybridisation in fixed cells. First, I will examine transcripts that are known to localise in other systems, for example b-actin transcripts that are present at the leading edge of migrating fibroblasts and neurites, and later, identify possible target transcripts expressed i n the selected cells using Drosophila cDNA microarrays. Once a reliable assay has been set up I will begin by inactivating genes already implicated in RNA localisation or microtubule-based transport, and screen more widely when high-throughput methods have been established. Genes that affect localisation without grossly disrupting cytoskeleton organisation will be analysed further genetically and biochemically to examine effects on RNA localisation and development. This RNAi screen will likely lead to the identification of a large number of genes involved in different pathways of RNA localisation and/or individual steps of these pathways. These will include genes already known to be involved in these pathways, but also new candidates. Since the molecular motors involved in RNA localisation are the same motors responsible for transport of other cargos, such as lipid vesicles, the project could contribute to a more integral view on intracellular transport.
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