REGULATION OF C-SRC PREMRNA SPLICING

C-SRC 前体 RNA 剪接的调控

• 批准号: 6018956
• 负责人: Douglas L Black
• 金额: $ 14万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018956
• 关键词: RNA binding protein; RNA splicing; cell differentiation; cell line; crosslink; gel mobility shift assay; genetic enhancer element; genetic regulatory element; introns; neurogenetics; neurons; nucleic acid sequence; precursor mRNA; protein binding; protein purification; protein structure; protein tyrosine kinase; protooncogene; small nuclear ribonucleoproteins; spliceosomes

Alternative splicing is an important means of genetic control in eucaryotic cells. By altering the splicing pattern of the primary transcript, a variety of proteins can be created from a single gene. Alternative splicing is especially prominent in the mammalian nervous system, where it regulates the production of many proteins that are important for neuronal development, function, and disease. Unfortunately, although our understanding of the general biochemistry of splicing has advanced significantly, much less is known of how splicing is regulated. The mouse c-src gene has provided an effective model system for studying a neuron-specific splicing event. Neurons produce a different form of the src protein from other tissues, resulting from the neuron-specific insertion of an extra exon (the N1 exon) into the src mRNA. Two major cis-acting elements that control N1 splicing have been identified by site specific mutagenesis of a transfected src mini-gene. These are an intronic splicing enhancer, downstream of N1, that activates splicing of the exon but is only partially neural specific, and the 3' splice site upstream of the exon that represses the splicing in non-neuronal cells. The combination of these two sequences confers neural specific splicing on a heterologous test exon. The regulated splicing of the N1 exon was reconstituted in extracts of neuronal and non-neuronal cells and some of the regulatory proteins have been identified. These include the KSRP, hnRNP F, hnRNP H and PTB proteins binding to the downstream enhancer, and the PTB protein also binding to the upstream 3' splice site. How the RNA/protein complexes at these sites combine to generate the precise tissue specific inclusion of an exon is still unclear. This project will pursue the molecular analysis of src neuron-specific splicing. Using a variety of biochemical assays, the assembly of the regulatory proteins onto the enhancer and repressor RNA sequences will be studied, and the interactions of these RNA/protein complexes with each other, and with components of the spliceosome will be dissected. New regulatory proteins will be characterized, including a neural specific form of PTB. The role of the recently described splicing enhancer protein, KSRP, will be studied and the functional domains of the protein delineated. Finally, simplified splicing systems will be developed for the analysis of individual regulatory proteins. Our goal is to understand in molecular detail how a simple change in splicing pattern is regulated in differentiated cells.

选择性剪接是一种重要的遗传控制手段 真核细胞。通过改变主节点的拼接模式 转录本，一个基因可以产生多种蛋白质。 选择性剪接在哺乳动物的神经细胞中尤为突出 系统，在那里它调节许多蛋白质的产生，这些蛋白质是 对神经元发育、功能和疾病很重要。 不幸的是，尽管我们对普通生物化学的理解 拼接技术已经有了显著的进步，但更少人知道它是如何 拼接是受监管的。小鼠c-src基因已经提供了一种有效的 用于研究神经元特定剪接事件的模型系统。神经元 从其他组织中产生不同形式的src蛋白， 由于神经元特异性地插入了额外的外显子(N1 外显子)整合到src基因中。控制N1的两个主要顺式作用元件 剪接是通过一种特定的位点突变来鉴定的 转染型src微型基因。这些是内含子剪接增强剂， 在N1下游，它激活外显子的剪接，但只是 部分神经元特异性，以及外显子上游的3‘剪接点 这抑制了非神经细胞的剪接。这两种技术的结合 这两个序列使神经特异性剪接在一个异源 检测外显子。N1外显子的调控剪接在 神经细胞和非神经细胞的提取物及其部分调节作用 蛋白质已经被鉴定出来。这些包括KSRP、hnRNP F、hnRNP 与下游增强子结合的H和PTB蛋白，以及PTB 蛋白质也与上游3‘剪接位点结合。怎么了？ 这些位点上的RNA/蛋白质复合体结合在一起，产生精确的 外显子的组织特异性包涵体仍不清楚。这个项目将 进行src神经元特异性剪接的分子分析。vbl.使用 各种生化检测，组装的调节蛋白 对增强子和抑制子RNA序列的研究，以及 这些RNA/蛋白质复合体之间的相互作用，以及与 剪接体的组件将被解剖。新的监管规定 将对蛋白质进行表征，包括一种神经特定形式的肺结核。 最近描述的剪接增强蛋白KSRP的作用将 并对该蛋白的功能结构域进行了研究。 最后，将开发用于分析的简化拼接系统 个体调节蛋白。我们的目标是了解 剪接模式中的简单变化是如何被调控的分子细节 在分化的细胞中。