Mechanisms of Signal-Induced Alternative Splicing: CD45

描述（由申请人提供）：本项目的最终目标是了解细胞外刺激通过信号诱导的选择性剪接过程调节细胞中蛋白质表达的机制。选择性剪接涉及新生前mRNA转录物内序列的差异连接，以在不同细胞类型或生长条件下形成不同的成熟mRNA。重要的是，这些差异剪接模式通常编码不同的蛋白质或改变顺式调节元件的存在，从而改变蛋白质表达水平。由于绝大多数人类基因经历某种形式的选择性剪接，该过程是人类蛋白质组内多样性和表达的主要决定因素。信号响应性选择性剪接用于控制蛋白质表达以响应细胞刺激，特别是在神经和免疫系统中，其中细胞必须对不断变化的环境条件做出强烈反应。人类CD 45基因是信号诱导的可变剪接的第一个已知例子之一，包含三个可变外显子，它们在响应免疫攻击时优先被跳过（抑制）。CD 45的这种受调节的剪接对于T细胞在免疫应答中的最佳功能是必不可少的，如CD 45剪接的误调节与人类自身免疫疾病之间的相关性所指示的。 此外，作为信号调节剪接的最佳表征的例子之一，CD 45是一个独特的有价值的模型来剖析可变剪接控制的机制。 CD 45外显子跳跃主要通过RNA结合蛋白hnRNP L在静息细胞中完成，其进一步通过相关的 蛋白PSF和hnRNP LL。 值得注意的是，由于与一种称为TRAP 150的特征不明显的蛋白质的磷酸化依赖性相互作用，PSF被抑制与静息细胞中的CD 45底物结合，为剪接因子的信号依赖性调节提供了新的范例。 目前的建议将利用这方面的知识，CD 45剪接阐明的分子机制，细胞活化调节选择性剪接在一个前所未有的细节水平。 具体而言，该建议旨在确定（1）hnRNP L/PSF/hnRNP LL如何改变剪接酶复合物（剪接体）的适当形成以阻断外显子包含，（2）hnRNP L和PSF影响剪接体的分子相互作用，以及（3）PSF的磷酸化如何控制其与TRAP 150的相互作用以调节PSF的功能。这些问题中的每一个都将通过生物化学方法来解决，以确定与调节活性相关的蛋白质-蛋白质，蛋白质-RNA和RNA-RNA相互作用，然后通过功能测定来确定这些相互作用的机制意义。这些问题的答案将产生一个完整的理解信号诱导的选择性剪接的生理学意义的例子的调节，并将提供新的见解，广泛地告知我们的理解的机制，这种调节可以发生。 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，从而适当地发挥功能并预防疾病状态。目前的研究表明，信号诱导的选择性剪接是调节细胞外刺激引起的蛋白质表达和细胞功能的重要机制。本提案中的研究旨在阐明细胞活化以前所未有的详细程度调节选择性剪接的分子机制，以预测基因突变的影响或操纵剪接用于治疗目的。

DESCRIPTION (provided by applicant): The ultimate goal of this project is to understand the mechanisms by which extracellular stimuli regulate protein expression in a cell through the process of signal-induced alternative splicing. Alternative splicing involves the differential joining of sequences within a nascent pre-mRNA transcript, to form distinct mature mRNAs in different cell types or growth conditions. Importantly, these differential splicing patterns typically encode distinct proteins or alter the presence of cis-regulatory elements thereby altering the level of protein expression. As the vast majority of human genes undergo some form of alternative splicing, this process is a major determinant of diversity and expression within the human proteome. Signal-responsive alternative splicing is used to control protein expression in response to cellular stimulation, particularly in the nervous and immune systems in which cells must react robustly to changing environmental conditions. The human CD45 gene, one of the first known examples of signal-induced alternative splicing, contains three variable exons that are preferentially skipped (repressed) in response to immune challenge. This regulated splicing of CD45 is essential for optimal function of T cells in an immune response as indicated by the correlation between mis-regulation of CD45 splicing and human autoimmune disease. Moreover, as one of the best-characterized examples of signal-regulated splicing, CD45 is a distinctively valuable model to dissect the mechanisms by which alternative splicing is controlled. CD45 exon skipping is primarily accomplished in resting cells through the RNA-binding protein hnRNP L, which is further joined by the related proteins PSF and hnRNP LL following cellular stimulation. Remarkably, PSF is inhibited from binding to the CD45 substrate in resting cells due to a phosphorylation-dependent interaction with a poorly characterized protein called TRAP150, providing a new paradigm for signal-dependent regulation of splicing factors. The current proposal will exploit this knowledge regarding CD45 splicing to elucidate the molecular mechanisms by which cellular activation regulates alternative splicing at an unprecedented level of detail. Specifically, this proposal seeks to determine (1) how appropriate formation of the splicing enzymatic complex (spliceosome) is altered by hnRNP L/PSF/hnRNP LL to block exon inclusion, (2) the molecular interactions through which hnRNP L and PSF influence the spliceosome and (3) how phosphorylation of PSF controls it's interaction with TRAP150 to regulate the function of PSF. Each of these questions will be addressed through biochemical methods to identify protein-protein, protein-RNA and RNA-RNA interactions that correlate with regulatory activity, followed by functional assays to determine the mechanistic significance of these interactions. Answers to these questions will yield a complete understanding of the regulation of a physiologically significant example of signal-induced alternative splicing, and will provide novel insight to broadly inform our understanding of the mechanisms by which such regulation can occur. 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 function appropriately and prevent disease states. Current studies demonstrate that an abundant mechanism for regulating protein expression and cellular function in response to extracellular stimulaton is that of signal-induced alternative splicing. The studies in this proposal seek to elucidate he molecular mechanisms by which cellular activation regulates alternative splicing at an unprecedented level of detail as required to predict the effect of genetic mutations or manipulate splicing for therapeutic purposes.