REGULATED SPLICING OF MESSENGER RNA PRECURSORS

本提案的主要目标是确定和描述 分子因子和事件，调节选择性剪接前 哺乳动物系统中的mRNA。 理解替代方案的重要性 拼接源于其在指定保真度方面的广泛作用， 多肽合成的多样性，因为这个过程是一个 转录后基因表达的重要控制点 水平 作为拟议研究重点的具体目标 旨在解决指定替代方案的机制， 外显子选择和选择性内含子保留的模型系统， 生物学上相关的，并且适合于实验分析。 One avenue 的研究将解决如何选择外显子的问题， 受到一个机制的积极监管， 模板导向的，外显子桥接的相互作用之间的基本 剪接因子U1 snRNP和U2 AF65。 这项工作是我们 先前使用大鼠前速激肽原的可变剪接研究 基因作为模型系统。 这项研究的一个重要目标是 用纯化的组分重建外显子桥接复合物， 用这个重组系统来表征分子间的相互作用 对功能很重要的基因（外显子选择）。 第二条大道 研究将探讨必要的剪接因子的作用， 替代内含子保留的对比机制，这涉及到一个 一个不寻常的小内含子嵌入在信使RNA中， 转录激活因子FosB。 小内含子的剪接导致 具有改变的功能特性的截短蛋白的表达。 顺式作用信号和反式作用组分的详细分析 将在体内和体外进行，以测试这一假设， 机制涉及剪接位点的失活或抑制 选择. 这项研究的一个重要目标是深入了解 选择内含子的剪接因子的不同作用 与外显子选择机制相比。 第三大道 研究将解决外显子的细胞特异性调节问题， 使用哺乳动物GABA A受体γ 2亚基基因进行选择。 在 在这个例子中，选择性剪接调节了24 各种脑组织中的核苷酸外显子。 的选择性剪接 γ 2亚基产生两种蛋白质同种型， 存在或不存在磷酸化位点， 在GABA A受体功能的调节中起着关键作用， 激酶。 拟议的研究将剖析序列元素 细胞特异性外显子选择所需的信息，并使用此信息 开发特定的检测方法来筛选调节分子。 一个重要 建议工作的重点和长期目标是隔离和 表征细胞特异性调节因子， 哺乳动物中枢神经系统中的选择性外显子选择。

The main objectives of this proposal are to identify and characterize the molecular factors and events that regulate alternative splicing of pre- mRNA in mammalian systems. The importance of understanding alternative splicing stems from its widespread role in specifying the fidelity and diversity of polypeptide synthesis and because this process is an important control point for gene expression at the post-transcriptional level. The specific aims that are the focus of the proposed research have been designed to address the mechanisms that specify alternative exon selection and alternative intron retention in model systems that are biologically relevant and amenable to experimental analysis. One avenue of research will address the question of how alternative exon selection is positively regulated by a mechanism that is proposed to involve template-directed, exon-bridging interactions between the essential splicing factors U1 snRNP and U2AF65. This work is a continuation of our previous studies of alternative splicing using the rat preprotachykinin gene as a model system. An important goal of this research is to reconstitute the exon-bridging complex with purified components and to use this reconstituted system to characterize the molecular interactions that are important for function (exon selection). A second avenue of research will explore the roles of essential splicing factors in the contrasting mechanism of alternative intron retention, which involves an unusual mini intron embedded in the messenger RNA encoding the transcriptional activator FosB. Splicing of the mini intron leads to the expression of a truncated protein with altered functional properties. A detailed analysis of cis-acting signals and trans-acting components will be carried out in vivo and in vitro to test the hypothesis that this mechanism involves a deactivation, or repression of splice site selection. An important goal of this research is to gain insight into the diverse roles of splicing factors that operate to select an intron in comparison to the mechanism of exon selection. The third avenue of research will address the question of cell-specific regulation of exon selection using the mammalian GABA A receptor gamma2 subunit gene. In this example, alternative splicing regulates the selection of a 24 nucleotide exon in various brain tissues. Alternative splicing of the gamma2 subunit gives rise to two protein isoforms that differ in the presence or absence of a phosphorylation site that is believed to play a pivotal role in the regulation of GABA A receptor function by protein kinases. The proposed research will dissect the sequence elements required for cell-specific exon selection and use this information to develop specific assays to screen for regulatory molecules. An important emphasis and long term goal of the proposed work is the isolation and characterization of cell-specific regulatory factors that direct alternative exon selection in the mammalian central nervous system.