The overall goal of this proposal is to elucidate the mechanisms involved in differential transport and localization of mRNA in eucaryotic cells. The experimental system under investigation is transport and localization of myelin basic protein (MBP) mRNA in oligodendrocytes - the cells that produce myelin in the central nervous system.
The specific aims are as follows: 1) isolate and characterize MBP mRNA granules from oligodendrocytes, using in vivo photochemical crosslinking and subcellular fractionation, 2) define transport and localization signals in MBP mRNA, using site-directed mutagenesis and microinjection, 3) identify factors in oligodendrocytes that are involved in transport and localization of MBP mRNA, using protein nucleic acid binding assays and advanced imaging techniques, 4) characterize the interaction between MBP and its own mRNA, using site-directed mutagenesis, in vitro evolution, and computer modelling, 5) visualize MBP mRNA transport and localization in intact nerve, using confocal laser scanning microscopy and advanced three dimensional visualization techniques, and 6) Design and test a synthetic RNA molecule that can take the place of MBP, using transgenic mice lacking this protein. The results of these experiments will provide a detailed picture of the molecular interactions involved in transport and localization of mRNA in eucaryotic cells. They may also lead to novel therapeutic approaches to demyelinating diseases such as multiple sclerosis and other autoimmune diseases that involve an immune response to a specific protein.
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