Regulation of C-SRC PreMRNA Splicing

描述（由申请人提供）：本项目的长期目标是从分子水平上了解mRNA剪接模式的简单变化如何在分化细胞中受到调节。初级基因转录物通常以替代模式剪接以从单个基因产生多种mRNA和蛋白质。选择性剪接在哺乳动物神经系统中特别常见，其中许多对神经元发育和活性重要的蛋白质通过剪接的变化以多种功能同种型表达。几种神经系统疾病，包括脊髓性肌萎缩症和额颞叶痴呆症的一种形式，都是由选择性剪接的错误和特定剪接异构体的错误产生引起的。尽管它在细胞分化、生理学和疾病中的重要性，但控制剪接位点选择的机制知之甚少。本工作将继续研究哺乳动物细胞c-src N1外显子的正调控和负调控。我们将把我们在这个相对简单的选择性剪接的例子中的发现与更复杂的系统联系起来。我们计划表征的结构和组装的多嘧啶道结合蛋白（PTB）复合物，抑制N1外显子剪接在非神经元细胞。我们将确定新的神经PTB蛋白在改变这种PTB复合物中的作用，并可能允许神经元细胞中N1剪接的去抑制。我们将分析N1外显子下游内含子剪接增强子的剪接刺激机制。最后，我们计划表征大的前mRNP复合物，包含一个迷你src前mRNA，并作为在体内和体外的剪接反应的基板。从这项工作中，我们希望精确地了解分子细节，各种组合输入如何决定剪接的变化。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand in molecular terms how a simple change in mRNA splicing pattern is regulated in differentiated cells. Primary gene transcripts are often spliced in alternative patterns to produce a variety of mRNAs and proteins from a single gene. Alternative splicing is especially common in the mammalian nervous system where many proteins important for neuronal development and activity are expressed in multiple functional isoforms through changes in splicing. Several neurologic diseases, including Spinal Muscular Atrophy and a form of Frontotemporal Dementia, arise from errors in alternative splicing and the misproduction of particular spliced isoforms. In spite of its importance in cell differentiation, physiology, and disease, the mechanisms controlling splice site choice are poorly understood. The proposed work will continue our analysis of the positive and negative regulation of the c-src N1 exon in mammalian cells. We will relate our findings on this relatively simple example of alternative splicing to more complicated systems. We plan to characterize the structure and assembly of the Polypyrimidine Tract Binding Protein (PTB) complex that represses N1 exon splicing in non-neuronal cells. We will determine the role of the new neural PTB protein in altering this PTB complex and perhaps allowing the derepression of N1 splicing in neuronal cells. We will analyze the mechanism of splicing stimulation by the intronic splicing enhancer downstream of the N1 exon. Finally, we plan to characterize the large pre-mRNP complexes that contain a mini src pre-mRNA and that serve as the substrate for the splicing reaction in vivo and in vitro. From this work we hope to understand in precise molecular detail how a variety of combinatorial inputs can determine a change in splicing.