Mechanisms of Signal-Induced Alternative Splicing: CD45

描述(申请人提供)：这个项目的长期目标是了解细胞外刺激如何通过信号诱导的选择性剪接过程诱导单个基因编码具有不同功能的蛋白质亚型。选择性剪接是人类蛋白质组多样性的主要决定因素，也是调节蛋白质表达的关键机制。特别是，越来越多的基因被证明对细胞外刺激做出了选择性剪接。此外，选择性剪接中的错误与许多人类疾病有关。尽管选择性剪接对人类健康具有重要意义，但人们对调控这一过程的机制知之甚少。这项提议试图理解CD45基因的受控剪接，它发生在T细胞激活的反应中，作为信号诱导选择性剪接调控的模型。这一建议的具体目的是：1)鉴定决定CD45剪接模式的顺式调控序列；2)鉴定和鉴定调控CD45剪接的核糖核酸结合蛋白；3)鉴定T细胞活化引起CD45剪接改变的途径的组成部分(S)。CD45调控的序列要求将通过分析CD45微基因内突变对CD45剪接的影响来确定。与CD45 RNA中功能元件结合的蛋白质将通过生化纯化来鉴定，体外和基于细胞的功能分析将被用来确定这些蛋白质调控CD45剪接的机制。最后，基于细胞的筛选将被用来鉴定和表征在激活诱导的选择性剪接方面缺乏的突变细胞，以确定从最初的信号事件到剪接调控的途径(S)。总之，这些研究将导致对激活信号通路导致选择性剪接调控的机制的更深入的了解，并在这样做的同时，可能为前mRNA剪接的治疗修饰提供潜在的靶点。

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.