Coordination of Inducible Alternative Splicing Networks in Human T Cells

描述(申请人提供)：替代前mRNA剪接是一种普遍存在的基因调控机制，允许精确控制由单个基因编码的功能不同的蛋白质亚型的表达。特别是，信号诱导的选择性剪接是一种强大的机制，细胞可以通过它来微调精确的蛋白质功能，以响应细胞环境的变化。在过去的一年里，一些研究表明，作为一种改变细胞功能以应对环境条件变化的手段，选择性剪接的广泛协调调节是响应细胞激活的一种手段。然而，协调信号诱导的多基因剪接的蛋白质和途径尚未得到全面的研究。最近，我们发现了一组广泛的基因，这些基因在T细胞激活时发生剪接变化，我们已经开始根据相似的调控模式、序列同源性和对特定RNA结合蛋白的敏感性对这些基因进行分组。在这个提案中，我们将扩展这些研究，以获得更完整的序列、蛋白质和机制，这些序列、蛋白质和机制协调调节T细胞激活时诱导的选择性剪接事件的程序。首先，我们将继续合作使用剪接微阵列来分析特定剪接调控蛋白的耗尽如何改变T细胞系中信号响应的替代剪接模式。我们还将利用生物信息学的方法来识别与我们识别的基因之间剪接调控的特定模式相关的序列基序。其次，我们将对剪接微基因进行系统的突变，以表征序列背景如何影响我们已经与激活诱导的选择性剪接相关的一个序列元件的功能。类似的方法也将被用来识别和/或验证控制信号诱导剪接的不同子集的额外调控序列。最后，我们将使用体外分析来鉴定和表征通过这些已识别的调控序列发挥功能的蛋白质。这些研究的最终目的是定义关于信号诱导的选择性剪接的基因的足够的机制信息，以便我们可以准确地预测在选择性剪接水平上对特定刺激做出反应的基因。这些研究也将对我们对组合剪接调控的理解产生广泛的影响，并提供对基本的、但缺乏特征的分子机制的详细见解，这些分子机制控制免疫系统的适当功能，对预防人类疾病至关重要。

DESCRIPTION (provided by applicant): Alternative pre-mRNA splicing is a ubiquitous mechanism of gene regulation that allows for precise control of the expression of functionally distinct protein isoforms encoded by a single gene. In particular, signal-induced alternative splicing is a powerful mechanism by which a cell can fine-tune precise protein function in response to changes in cellular environment. Several studies in the past year have demonstrated widespread coordinate regulation of alternative splicing in response to cellular activation as a means to alter cellular function in response to changing environmental conditions. However, the proteins and pathways that coordinate the signal-induced splicing of multiple genes has not yet been investigated in a comprehensive manner. Recently we identified a broad set of genes that undergo changes in splicing in response to T cell activation, and we have begun to group these genes based on similar patterns of regulation, sequence homology, and sensitivity to specific RNA-binding proteins. In this proposal we will extend these studies to gain a more complete understanding of the sequences, proteins and mechanisms that coordinately regulate the program of alternative splicing events that are induced upon T cell activation. First, we will continue our collaborative use of splicing microarrays to analyze how depletion of specific splicing regulatory proteins alters the signal-responsive alternative splicing profile in a T cell line. We will also utilize a bioinformatics approach to identify sequence motifs that correlate with specific patterns of splicing regulation among the genes we identify. Secondly, we will perform systematic mutagenesis of splicing minigenes to characterize how sequence context influences the function of the one sequence element that we have already correlated with activation-induced alternative splicing. A similar approach will also be used to identify and/or validate additional regulatory sequences that control distinct subsets of signal-induced splicing. Finally, we will use in vitro assays to identify and characterize the proteins that function via these identified regulatory sequences. An ultimate goal of these research aims is to define sufficient mechanistic information regarding genes that undergo signal-induced alternative splicing, that we may accurately predict genes which are regulated at the level of alternative splicing in response to a particular stimuli. These studies will also have a broad impact on our understanding of combinatorial splicing regulation, as well as provide detailed insight into essential, but poorly characterized, molecular mechanisms that control proper function of the immune system and are critical to the prevention of human disease.