Mechanisms of Signal-Induced Alternative Splicing: CD45

描述(申请人提供)：这个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内蛋白质表达的机制。选择性剪接涉及新生前mRNA转录本内序列的差异连接，以在不同的细胞类型或生长条件下形成不同的成熟mRNAs。重要的是，这些不同的剪接模式通常编码不同的蛋白质或改变顺式调控元件的存在，从而改变蛋白质的表达水平。由于绝大多数人类基因经历了某种形式的选择性剪接，这一过程是人类蛋白质组中多样性和表达的主要决定因素。信号响应性选择性剪接被用来控制蛋白质的表达以响应细胞刺激，特别是在神经和免疫系统中，在这些系统中，细胞必须对不断变化的环境条件做出强有力的反应。人类CD45基因是已知的最早的信号诱导选择性剪接的例子之一，它包含三个可变外显子，它们在免疫攻击时优先被跳过(抑制)。这种受调控的CD45剪接对于T细胞在免疫反应中的最佳功能是必不可少的，CD45剪接的错误调控与人类自身免疫性疾病之间的相关性表明了这一点。此外，作为信号调控剪接最典型的例子之一，CD45是分析选择性剪接调控机制的一个有独特价值的模型。CD45外显子的跳跃主要是通过rna结合蛋白hnRNP L在静息细胞中完成的，该蛋白还与相关的 细胞刺激后的蛋白质PSF和hnRNP L1。值得注意的是，由于PSF与一种特性不佳的蛋白质TRAP150的磷酸化依赖的相互作用，PSF被抑制与静息细胞中的CD45底物结合，这为信号依赖的剪接因子调控提供了一种新的范式。目前的提议将利用这一关于CD45剪接的知识来阐明细胞激活以前所未有的细节水平调控选择性剪接的分子机制。具体地说，本建议试图确定(1)hnRNP L/pSF/hnRNP 11如何改变剪接酶复合体(剪接体)的适当形成以阻断外显子夹杂，(2)hnRNP L和pSF通过什么分子相互作用影响剪接体，以及(3)pSF的磷酸化如何控制它与TRAP150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化方法来解决，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，然后通过功能分析来确定这些相互作用的机制意义。对这些问题的回答将使我们对信号诱导的选择性剪接的生理意义的例子有一个完整的理解，并将提供新的见解来广泛地了解这种调节发生的机制。

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.