The growth-associated protein, GAP-43, is a neuronal phosphoprotein that is required for the initial establishment and remodeling of neural connections. Work done under our previous grant has demonstrated that GAP-43 gene expression is post- transcriptionally regulated by selective changes in the stability of its mRNA, and that this process depends on the interaction of a highly conserved regulatory element in the mRNA with neuronal- specific RNA-binding proteins. We have recently identified one of these proteins as HuD, the mammalian equivalent of Elav, an RNA-binding protein that is critical for nervous system development in Drosophila. Based upon our preliminary studies, we propose that HuD protects that GAP-43 mRNA against ribonuclease attack, leading to an increase in gene expression and neurite outgrowth. To test this hypothesis, we plan to investigate the mechanism by which HuD controls GAP-43 gene expression at the molecular (Aim 1), cellular (Aim 2), and in vivo levels (Aim 3): Aim 1: To study the molecular mechanism by which the RNA-binding protein HuD stabilizes the GAP-43 mRNA. We will use a combination of cell-free mRNA decay assays and RNA- binding studies to examine whether binding of HuD to the GAP-43 mRNA protects this from deadenylation. Aim 2. To determine the cellular specificity of the effect of HuD on GAP-43 gene expression and neurite outgrowth. These studies will utilize primary neuronal cultures, ES cells and neural cell lines. Aim 3: To use genetic manipulation techniques to investigate the relationship between HuD and GAP-43 in vivo. We will make use of transgenic mice and viral vectors to probe HuD's function in vivo. The proposed studies will characterize the role of HuD in the post-transcriptional regulation of the GAP-43 gene. Given the role of GAP-43 in nervous system development and regeneration, the elucidation of regulatory mechanisms controlling its expression have potential applications for the treatment of a broad range of conditions from neurodevelopmental disorders, to brain trauma and spinal cord injury.
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