REGULATION OF C-SRC PREMRNA SPLICING

选择性剪接是一种重要的遗传控制手段， 真核细胞 通过改变主细胞的拼接模式 转录本，多种蛋白质可以从一个单一的基因创建。 选择性剪接在哺乳动物的神经系统中尤为突出。 系统，在那里它调节许多蛋白质的产生， 对神经元发育、功能和疾病都很重要。 不幸的是，尽管我们对普通生物化学的理解 拼接的技术已经有了很大的进步，但人们对如何拼接知之甚少。 剪接受到调节。小鼠c-src基因提供了有效的 用于研究神经元特异性剪接事件的模型系统。 神经元 产生与其他组织不同形式的src蛋白， 这是由于神经元特异性插入一个额外的外显子（N1 外显子）插入src mRNA中。 控制N1的两个主要顺式作用元件 剪接已经通过对一个突变位点的位点特异性诱变来鉴定， 转染src mini-gene。 这些是内含子剪接增强子 N1的下游，激活外显子的剪接，但仅 部分神经特异性，外显子上游的3'剪接位点 抑制非神经元细胞的剪接。的组合 这两个序列赋予神经特异性剪接在异源 测试外显子。 N1外显子的受调控剪接被重建， 神经元和非神经元细胞的提取物以及一些调节性的 蛋白质已被鉴定。 其中包括KSRP、hnRNP F、hnRNP H和PTB蛋白结合到下游增强子，并且PTB 蛋白质也结合到上游3'剪接位点。 如何 这些位点上的RNA/蛋白质复合物联合收割机产生精确的 外显子的组织特异性包含仍不清楚。该项目将 进行src神经元特异性剪接的分子分析。 使用 各种生化分析，调节蛋白的组装 将研究增强子和阻遏子RNA序列， 这些RNA/蛋白质复合物彼此之间的相互作用，以及 剪接体的组分将被解剖。 新的监管 蛋白质将被表征，包括PTB的神经特异性形式。 最近描述的剪接增强子蛋白KSRP的作用， 研究和蛋白质的功能域划定。 最后，将开发用于分析的简化拼接系统 单个调节蛋白。我们的目标是了解 剪接模式的简单变化是如何被调控的分子细节 在分化的细胞中。

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.