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Molecular mechanims of mRNA transport in neurons

Final Report Summary - NEURORNATRANSPORT (Molecular mechanims of mRNA transport in neurons)

Transport of mRNAs into defined subcellular compartments coupled to their local translation is a powerful mechanism to control gene expression in time and space. Originally believed to be a rare process involving only a handful of transcripts in highly specialised cells, more recent evidence suggests that potentially hundreds of transcripts are localised to specific subcellular compartments in a wide range of cell types. Moreover, mRNA localisation has been shown to be functionally important for diverse cell biological processes, such as cell polarity, cell migration, asymmetric cell division and signal transduction. However, the molecular mechanism governing mRNA localisation is currently unclear. In this project we aim to shed light on the regulation and functional significance of mRNA transport using the genetically tractable organism Drosophila melanogaster as a model system.
We have developed a sensitive protocol that allows detection of mRNPs in the intact nervous system of the Drosophila larva. We have used this assay to conduct a screen in fly sensory neurons of 300 genomic 3’UTRs for dendritic or axonal localisation signals. We found that although low level dendritic localisation is widespread, efficient localisation into dendrites is a rare process. We identified a single 3’UTR that harbours a strong dendritic localisation signal. We have mapped a region within this 3’UTR that is absolutely required for dendritic localisation and now use this information to test for the functional significance of the process by genome engineering. To this end we have developed a powerful toolbox for CRISPR/Cas genome engineering in Drosophila. The toolbox consists of transgenic Cas9 fly strains and high efficiency gRNA vectors. In a proof-of-principle study we have demonstrated that these reagents allow genome engineering with remarkable high efficiency, including precise modifications of the genome by homology-directed repair. We now apply this technology to study in detail dendritic mRNA localisation of our candidate gene