Mechanisms and consequences of CELF2 regulation in T cell development

描述(申请人提供)：本提案的最终目标是了解替代剪接是如何控制细胞发育的。选择性剪接涉及从特定基因产生不同的蛋白质编码信息。因此，选择性剪接在产生蛋白质多样性和调节蛋白质表达方面发挥着重要而普遍的作用。特别是，在发育过程中或在对环境提示的反应中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)通过一系列由配体依赖的信号通路触发的检查点进行发育。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。众所周知，配体诱导的胸腺细胞信号会引起转录谱的变化，然而，令人惊讶的是，胸腺细胞中选择性剪接的调节在很大程度上还没有被探索。作为纠正这种知识匮乏的第一步，最近的数据表明，在胸腺发育过程中剪接调节蛋白CELF2的表达增加，调节编码转录因子LEF1的基因的选择性剪接。LEF1剪接的调节反过来又有助于刺激T细胞受体(TCR)的表达，a T细胞成熟和功能的必要步骤。这项建议旨在建立在这一初始数据的基础上 确定胸腺发育检查点对CELF2的调节机制(S)，以及在T细胞成熟过程中，CELF2如何反过来调节特定的选择性剪接事件。具体地说，这项建议的目的是(1)通过检测细胞刺激诱导的mRNA稳定性、转录变化和翻译后修饰来确定CELF2中导致信号依赖性表达和活性增加的确切分子变化，(2)利用生化分析来揭示CELF2调节剪接的分子机制以及这种机制是如何以信号依赖的方式发生变化的，以及(3)利用新兴的高通量技术来获得胸腺细胞中CELF2 RNA结合和功能的全局视角。总之，这些研究将为胸腺细胞发育中的不同剪接程序提供前所未有的洞察力，这是朝着确定剪接调控在塑造功能免疫系统中的广泛作用迈出的关键一步。此外，由于CELF2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的影响。此外，这些研究将增加我们对发育调控剪接的机制以及信号通路和剪接调控蛋白之间的联系的总体理解。

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.