Mechanisms of Signal-Induced Alternative Splicing: CD45

信号诱导的选择性剪接机制：CD45

• 批准号: 7052874
• 负责人: KRISTEN W LYNCH
• 金额: $ 24.66万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7052874
• 关键词: CD antigens; RNA binding protein; RNA splicing; T lymphocyte; biological signal transduction; cell line; fusion gene; genetic regulatory element; leukocyte activation /transformation; mutant; nucleic acid sequence; posttranslational modifications; protein isoforms; protein purification; protein quantitation /detection; protein structure function; protein tyrosine phosphatase

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how extracellular stimuli can induce a single gene to encode functionally distinct protein isoforms through the process of signaling induced alternative splicing. Alternative splicing is a major determinant of diversity within the human proteome and is a critical mechanism for modulating protein expression. In particular, an increasing number of genes have been shown to undergo alternative splicing in response to extracellular stimuli. Moreover, mistakes in alternative splicing have been linked to numerous human diseases. Despite the significance of alternative splicing to human health, relatively little is understood about the mechanisms that regulate this process. This proposal seeks to understand the regulated splicing of the CD45 gene that occurs in response to T cell activation, as a model for signaling-induced regulation of alternative splicing. The Specific Aims of this proposal are 1) To identify the cis-regulatory sequences that determine the pattern of CD45 splicing, 2) To identify and characterize the RNA-binding proteins that regulate CD45 splicing, and 3) To identify components of the pathway(s) by which T cell activation induces changes in CD45 splicing. The sequence requirements for CD45 regulation will be determined by assaying the effects of mutations within CD45 minigenes on the splicing of CD45. Protein that bind to the functional elements within CD45 RNA will be identified by biochemical purification, and both in vitro and cell-based functional assays will be used to determine the mechanisms by which these proteins regulate CD45 splicing. Finally, a cell-based screen will be used to identify and characterize mutant cells that are deficient in activation-induced alternative splicing, in order to determine the pathway(s) that lead from the initial signaling event to the regulation of splicing. Together these studies will lead to greater insight as to the mechanisms by which activation of signaling pathways leads to regulation of alternative splicing, and in so doing, are likely to suggest potential targets for therapeutic modification of pre-mRNA splicing.

描述（由申请人提供）：该项目的长期目标是了解细胞外刺激如何通过信号传导诱导的替代剪接过程来诱导单个基因编码功能上不同的蛋白质同工型。替代剪接是人类蛋白质组中多样性的主要决定因素，是调节蛋白质表达的关键机制。特别是，越来越多的基因已显示出对细胞外刺激的替代剪接。此外，替代剪接中的错误与许多人类疾病有关。尽管替代剪接对人类健康的重要性，但对调节该过程的机制的理解很少。该提案试图了解响应T细胞激活而发生的CD45基因的调节剪接，作为信号传导诱导的替代剪接调节的模型。该提案的具体目的是1）确定确定CD45拼接模式的顺式调节序列，2）识别和表征调节CD45拼接的RNA结合蛋白，以及3）以识别T细胞活化的途径成分，从而诱导CD45剪接的变化。 CD45调控的序列要求将通过分析CD45微型烯内突变对CD45剪接的影响确定。与CD45 RNA中功能元件结合的蛋白质将通过生化纯化鉴定，并且在体外和基于细胞的功能测定中都将用于确定这些蛋白调节CD45剪接的机制。最后，基于细胞的屏幕将用于识别和表征不足于激活诱导的替代剪接的突变细胞，以确定从初始信号事件到剪接调节的途径。这些研究共同对信号通路的激活导致替代剪接的调节的机制有更大的见解，而这样做可能会暗示潜在的靶标，以修饰MRNA前剪接的治疗性修饰。