Mechanisms and consequences of CELF2 regulation in T cell development

描述（由申请人提供）：本提案的最终目标是了解选择性剪接如何控制细胞发育。 选择性剪接涉及来自给定基因的不同蛋白质编码信息的差异产生。 因此，选择性剪接在产生蛋白质多样性和调节蛋白质表达中起着重要和普遍的作用。特别是，在发育过程中或响应环境线索时发生的剪接模式的变化有助于确定细胞的功能反应或分化途径。 T细胞的发育为研究诱导的选择性剪接的机制和功能后果提供了一个特别丰富的系统，因为未成熟的T细胞（胸腺细胞）通过配体依赖性信号通路触发的一系列检查点在发育过程中进展。重要的是，这些检查点的缺陷会导致免疫缺陷或自身免疫性疾病。胸腺细胞中配体诱导的信号传导已知会诱导转录谱的变化，然而，引人注目的是，胸腺细胞中选择性剪接的调节在很大程度上尚未探索。作为纠正这种知识缺乏的第一步，最近的数据表明，增加表达的剪接调节蛋白CELF 2在胸腺发育调节选择性剪接的基因编码的转录因子LEF 1。LEF1剪接的调节反过来有助于T细胞受体（TCR）的刺激表达， 这是T细胞成熟和功能的必要步骤。本提案力求在这一初步数据的基础上 确定CELF 2响应胸腺发育检查点而调节的机制，以及这反过来如何调节T细胞成熟过程中的特定选择性剪接事件。 具体而言，该提议的目的是（1）通过检查细胞刺激诱导的mRNA稳定性、转录变化和翻译后修饰，确定CELF 2中导致信号依赖性表达和活性增加的精确分子变化，（2）利用生物化学测定来揭示CELF 2调节剪接的分子机制以及这种机制如何以信号依赖性方式变化，以及（3）利用新兴的高表达的细胞因子，通量技术，以获得胸腺细胞中CELF 2 RNA结合和功能的全局视图。这些研究将为胸腺细胞发育中的选择性剪接程序提供前所未有的见解，这是确定剪接调节在塑造功能性免疫系统中的广泛作用的重要一步。此外，由于CELF 2的错误调节与心脏，肌肉和胸腺的发育缺陷和疾病有关，这些研究对人类健康有直接影响。此外，这些研究将增加我们对发育调控剪接机制以及信号通路和剪接调控蛋白之间联系的总体理解。 公共卫生相关性：所有的人类细胞都必须能够快速而精确地改变它们的蛋白质表达，以响应给定的刺激，从而通过发育检查点，适当地发挥功能并预防疾病状态。特别是，主动免疫系统的发展涉及蛋白质表达变化的复杂程序。该提案中的研究调查了一种新发现的在免疫系统发育过程中控制蛋白质表达的机制，从而为蛋白质表达机制提供了前所未有的见解，通过这种机制可以控制免疫系统的活性。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to understand how alternative splicing controls cellular development. Alternative splicing involves the differential production of distinct protein-coding messages from a given gene. As such alternative splicing plays an essential and ubiquitous role in generating protein diversity and regulating protein expression. In particular, changes in splicing patterns that occur during development or in response to environmental cues help determine the functional response or differentiation pathway of a cell. The development of T cells provides an especiall rich system for studying the mechanisms and functional consequences of induced alternative splicing, as immature T cells (thymocytes) progress through development via a series of check-points triggered by ligand-dependent signaling pathways. Importantly, defects in these check-points result in immunodeficiencies or autoimmune diseases. Ligand-induced signaling in thymocytes is known to induce changes in transcription profiles~ however, strikingly, the regulation of alternative splicing in thymocytes has been largely unexplored. As a first step toward correcting this dearth of knowledge, recent data has demonstrated that increased expression of the splicing regulatory protein CELF2 during thymic development regulates alternative splicing of the gene encoding the transcription factor LEF1. The regulation of LEF1 splicing, in turn, contributes to the stimulated expression of the T cell receptor (TCR), a necessary step in T cell maturation and function. This proposal seeks to build on this initial data to determine the mechanism(s) by which CELF2 is regulated in response to developmental check-points in the thymus and how this in turn regulates specific alternative splicing events during T cell maturation. Specifically, the aims of this proposal are to (1) determine the precise molecular changes in CELF2 that lead to signal-dependent increases in expression and activity by examining mRNA stability, transcription changes and post-translational modifications induced by cell stimulation, (2) utilize biochemical assays to uncover the molecular mechanisms by which CELF2 regulates splicing and how this changes in a signal-dependent manner and (3) exploit emerging high- throughput technologies to gain a global view of CELF2 RNA binding and function in thymocytes. Together these studies will provide unprecedented insight into alternative splicing programs in developing thymocytes, an essential step toward determining the broad role of splicing regulation in the shaping of a functional immune system. Furthermore, as mis-regulation of CELF2 has been linked to developmental defects and disease of heart, muscle and thymus these studies have direct implications for human health. In addition, these studies will increase our general understanding of mechanisms of developmentally regulated splicing and the connection between signaling pathways and splicing regulatory proteins. PUBLIC HEALTH RELEVANCE: All human cells must be able to alter their protein expression rapidly and precisely in response to a given stimuli in order to progress through developmental check points, function appropriately and prevent disease states. In particular, development of an active immune system involves a complex program of changes in protein expression. The studies in this proposal investigate a newly identified mechanism for controlling protein expression during the development of the immune system, thereby providing unprecedented insight into mechanisms of protein expression through which the activity of the immune system can be controlled.