The expression of the growth-associated protein GAP-43 is critical for the proper establishment of neuronal connections. Given the significance of GAP-43 to normal brain development and the existence of human disorders associated with abnormal levels of this protein, it is extremely important to define the mechanisms controlling the expression of the GAP-43 gene. We have previously shown that GAP-43 gene expression is controlled by changes in the stability of its mRNA. Furthermore, we found that this process is mediated by the interaction of the highly conserved 3'untranslated region (3'UTR) and neural-specific RNA-binding proteins. Subsequent mapping of these sequences demonstrated that the element responsible for the phorbol ester-induced stabilization of the mRNA also contains recognition sites for the neuronal RNA-binding protein HuD. Based upon these findings, our hypothesis is that RNA-protein interactions between specific GAP-43 3' UTR sequences and RNA-binding proteins control GAP-43 gene expression during neural differentiation. To test this idea, we propose the following Specific Aims: 1. To define the sequences responsible for the TPA-induced stabilization of the GAP-43 mRNA. To map these sequences, we will generate specific GAP-43 3' UTR deletion mutants and chimeras with the beta-globin gene. Since HuD's binding site is localized in the same 3' UTR fragment that confers responsiveness to TPA, we will study the effect of selected deletions and point mutations at this site on GAP-43 mRNA stability. Finally, to gain further insights into the mechanisms of degradation of this mRNA, we will determine the rate of deadenylation of several GAP-43 3' UTR constructs in control and TPA-induced cells. 2. To study the role of HuD and other GAP-43 mRNA binding proteins on the rate of degradation of this mRNA. RNA-protein interactions and GAP-43 mRNA stability will be analyzed in parallel in vitro, in a polysome-based mRNA decay system and in permanently transfected cells. HuD and other specific factors will be extracted from polysomes by salt washes followed by treatment of the extracts by poly(U)-Sepharose or precipitation with specific 3. To examine the mechanisms that control the stability of the GAP-43 and other neural mRNAs using exogenous 3'UTR sequences as competitors. Transfected PC12 cells will be used to examine the effect of exogenous 3' UTR sequences on the expression of the endogenous GAP-43 mRNA. These studies will complement those preformed by Dr Rachel Neve using HSV-1 vectors to deliver constructs containing the GAP-43 3' UTR to neurons in primary cultures and in vivo. The proposed studies will provide novel information on the mechanisms that control the expression of the GAP-43 gene during neural development. Given that other important growth-associated genes are also regulated by mRNA stability mechanisms, it is conceivable that the cis-and trans-regulators of GAP-43 gene expression identified here also contribute to regulate the coordinated expression of tissue-specific genes during the development of the nervous system.
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