MECHANISMS OF CONTROL OF THE GAP-43 GENE

生长相关蛋白GAP-43是一种神经元磷酸蛋白，在神经连接的初始建立和重塑过程中是必需的。在我们之前的资助下所做的工作已经证明，GAP-43基因的表达是由其mRNA稳定性的选择性变化在转录后调节的，并且这一过程依赖于mRNA中一个高度保守的调节元件与神经元特异性RNA结合蛋白的相互作用。我们最近鉴定了其中一种蛋白质HUD，它是哺乳动物中相当于Elav的蛋白质，是一种RNA结合蛋白，对果蝇的神经系统发育至关重要。根据我们的初步研究，我们认为HUD保护GAP-43mRNA免受核糖核酸酶的攻击，导致基因表达增加和轴突生长。为了验证这一假说，我们计划从分子(目标1)、细胞(目标2)和体内(目标3)水平研究HUD控制GAP-43基因表达的机制：目的1：研究RNA结合蛋白HUD稳定GAP-43mRNA的分子机制。我们将使用无细胞mRNA衰变分析和RNA结合研究相结合的方法来检查HUD与GAP-43 mRNA的结合是否能保护其免于去烯化。目的2.确定HUD对GAP-43基因表达和神经突起生长影响的细胞特异性。这些研究将利用原代神经元培养、ES细胞和神经细胞系。目的：利用基因操作技术，在体内研究HUD与GAP-43的关系。我们将利用转基因小鼠和病毒载体来检测HUD在体内的功能。拟议的研究将表征HUD在GAP-43基因转录后调控中的作用。鉴于GAP-43在神经系统发育和再生中的作用，阐明其表达的调控机制在治疗从神经发育障碍到脑创伤和脊髓损伤的广泛疾病中具有潜在的应用价值。

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.