Molecular Mechanisms and Signal-Induced Regulation of Alternative Splicing

 描述(申请人提供)：这个项目的最终目标是了解RNA加工由信号通路控制的分子机制，以及这一调节对细胞功能的影响。本供资期间的具体重点是 T细胞活化过程中前mRNA选择性剪接的调控。备用剪接是一种 基本的和普遍存在的基因调控机制，允许在不同的细胞类型或环境条件下多样化和控制蛋白质的表达。特别是，信号诱导的选择性剪接在神经元活动、营养感知、肿瘤发生和免疫功能中普遍存在。然而，关于信号通路如何控制选择性剪接的机械论观点仍然缺乏。T细胞激活为复杂的细胞反应提供了一个很好的模型系统，因为多个信号通路在抗原结合的下游被触发，并单独地和协同地作用于诱导T细胞效应器功能。重要的是，已知数百个基因在T细胞激活后发生选择性剪接；然而，这些基因是如何调节的，哪些基因是由重叠的机制/途径共同调节的，以及这种调节的最终功能结果是什么，仍有待确定。这一建议将通过利用最近发展的方法和系统来解决这些悬而未决的问题，以确定：(1)导致T细胞抗原刺激后选择性剪接变化的特定信号通路，以及将每个信号通路与各自的剪接靶标连接起来的RNA结合蛋白(RNAbindingProteins，RBPs)；(2)RBPs和/或细胞信号调节剪接体组装中特定过渡到直接异构体表达的分子机制；(3)选择性剪接通过调节相关激酶的异构体表达而对细胞信号的功能影响；以及(4)选择性剪接的调节如何与其他RNA生物发生事件协调，如转录和3‘端加工。总之，这些研究将为在T细胞激活过程中信号和剪接在塑造细胞功能中的相互作用提供新的见解。由于T细胞激活时诱导的信号通路也在与细胞生长、代谢和癌症相关的通路中发挥作用，因此这些研究获得的见解将广泛适用于远远超出免疫系统的许多细胞反应。此外，这里提出的研究将揭示细胞调节剪接体组装的分子机制，以及限制性商业惯例如何协调控制剪接与RNA加工中的其他步骤。因此，这些实验的结果将显著增加对控制选择性剪接的机制的理解，选择性剪接是调节所有人类细胞类型的基因表达的一个基本和普遍的过程。

 DESCRIPTION (provided by applicant): The ultimate goal of this project is to understand the molecular mechanisms by which RNA processing is controlled by signaling pathways, and the impact of this regulation on cellular function. The specific focus of the current funding period is on regulation of alternative pre-mRNA splicing during T cell activation. Alternative splicing is an essential and ubiquitous mechanism of gene regulation, which allows for the diversification and control of protein expression in distinct cell-types or environmental conditions. In particular, itis well established that signal-induced alternative splicing is pervasive during neuronal activity, nutrient sensing, oncogenesis and immune function. However, a mechanistic view of how signaling pathways control alternative splicing remains lacking. T cell activation provides an excellent model system for complex cellular responses, in that multiple signaling pathways are triggered downstream of antigen engagement and act, individually and cooperatively, to induce T cell effector functions. Importantly, several hundred genes are known to undergo alternative splicing in response to T cell activation; however, it remains to be determined how these genes are regulated, which genes are co-regulated by overlapping mechanisms/pathways, and what the ultimate functional consequence is of such regulation. This proposal will address these unanswered questions of signal- induced alternative splicing by leveraging recently developed methodologies and systems to determine: (1) the specific signaling pathways that lead to changes in alternative splicing following antigen stimulation of T cells, and the RNA binding proteins (RBPs) that connect each signaling pathway with their respective splicing targets; (2) the molecular mechanisms by which RBPs and/or cell signaling regulate specific transitions in spliceosome assembly to direct isoform expression; (3) the functional impact of alternative splicing on cell signaling through regulating isoform expression of related kinases; and (4) how regulation of alternative splicing is coordinated with other RNA biogenesis events such as transcription and 3' end processing. Together these studies will provide novel insight regarding the interplay of signaling and splicing in shaping cellular function during T cell activation. Sinc the signaling pathways induced upon T cell activation are also functional in pathways related to cell growth, metabolism and cancer, the insight gained in these studies will be broadly applicable to numerous cellular responses far beyond the immune system. In addition, the studies proposed here will reveal new paradigms regarding the molecular mechanisms by which cells regulate spliceosome assembly, and how RBPs coordinately control splicing along with other steps in RNA processing. Results from these experiments will thus significantly increase understanding of the mechanisms that control alternative splicing, an essential and ubiquitous process for regulating gene expression across all human cell types.