REGULATED SPLICING OF MESSENGER RNA PRECURSORS

信使 RNA 前体的调控剪接

• 批准号: 3296801
• 负责人: PAULA J GRABOWSKI
• 金额: $ 18.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296801
• 关键词: GABA receptor; RNA binding protein; RNA splicing; brain cell; cell type; crosslink; gene expression; genetic regulatory element; intermolecular interaction; molecular cloning; nucleic acid probes; nucleic acid sequence; phosphorylation; plasmids; precursor mRNA; protein reconstitution; small nuclear ribonucleoproteins; tissue /cell culture; transcription factor

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.

这项提案的主要目标是确定和描述 调控Pre-2选择性剪接的分子因素和事件 在哺乳动物系统中的mRNA。了解替代方案的重要性 拼接源于它在指定保真度和 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。作为拟议研究的重点的具体目标 旨在解决指定替代方案的机制 模型系统中的外显子选择和替代内含子保留 生物学上相关的，易于实验分析的。一条大道 研究将解决如何选择替代外显子的问题 受到一种机制的积极监管，该机制被提议涉及 以模板为导向的、外显子桥接的基本基因之间的相互作用 剪接因子U1、SnRNP和U2AF65。这项工作是我们工作的延续 大鼠快速原激肽原选择性剪接的研究进展 基因作为一个模型系统。这项研究的一个重要目标是 用纯化的组分重组外显子桥联复合体并 用这个重组的系统来表征分子间的相互作用 对功能(外显子选择)很重要的基因。第二条道路 研究将探索基本剪接因子在 另一种内含子保留的对比机制，涉及一个 不同寻常的微小内含子嵌入信使RNA编码 转录激活因子FosB。微小内含子的拼接导致 功能性质改变的截短蛋白的表达。 对顺式作用信号和反式作用成分的详细分析 将在体内和体外进行，以检验这一假设 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 剪接因子在选择内含子中的不同作用 与外显子选择的机制相比较。第三条大道 研究将解决外显子的细胞特异性调控问题 利用哺乳动物GABAA受体Gamma2亚单位基因进行选择。在……里面 本例中，备用剪接控制24的选择 不同脑组织中的核苷酸外显子。可选的拼接 Gamma2亚基产生两种不同的蛋白质亚型 存在或不存在被认为起作用的磷酸化位点 蛋白质在调节GABA受体功能中的关键作用 激活剂。拟议的研究将对序列元素进行剖析 细胞特定外显子选择所需的信息，并使用该信息 开发特定的分析方法来筛选调节分子。一个重要的 拟议工作的重点和长期目标是孤立和 细胞特异性调控因子的特性 哺乳动物中枢神经系统中的选择性外显子选择。