Mechanisms of Signal-Induced Alternative Splicing: CD45

描述（由申请人提供）：该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。选择性剪接涉及在新生的pre-mRNA转录物中序列的差异连接，以在不同的细胞类型或生长条件下形成不同的成熟mrna。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，这一过程是人类蛋白质组多样性和表达的主要决定因素。信号响应选择性剪接用于控制细胞刺激反应中的蛋白质表达，特别是在神经和免疫系统中，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基因是最早已知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，在应答免疫挑战时被优先跳过（抑制）。CD45剪接的调节对于T细胞在免疫应答中的最佳功能至关重要，CD45剪接的错误调节与人类自身免疫性疾病之间的相关性表明了这一点。此外，作为信号调节剪接的最典型的例子之一，CD45是一个独特的有价值的模型，可以分析控制选择性剪接的机制。CD45外显子跳变主要是在静息细胞中通过rna结合蛋白hnRNP L完成的，该蛋白进一步通过相关基因的加入

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.