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。选择性剪接涉及从给定基因中产生不同蛋白质编码信息的差异。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起着必不可少的作用。特别是，剪接模式的变化发生在发育过程中或响应环境线索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性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，胸腺细胞中配体诱导的信号传导可诱导转录谱的变化，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被探索。作为纠正这一知识匮乏的第一步，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，可以调节编码转录因子LEF1的基因的选择性剪接。反过来，调节LEF1剪接有助于刺激T细胞受体（TCR）的表达

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.