Mechanisms of Signal-Induced Alternative Splicing: CD45

描述（由申请人提供）：该项目的长期目标是了解细胞外刺激如何通过信号诱导的选择性剪接过程诱导单个基因编码功能不同的蛋白质异构体。选择性剪接是人类蛋白质组多样性的主要决定因素，是调节蛋白质表达的关键机制。越来越多的基因已经被证明在细胞外刺激下进行选择性剪接，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。此外，选择性剪接中的错误，包括抑制信号诱导的剪接调节，与许多人类疾病有关。然而，对调节这一过程的蛋白质和机制的了解相对较少。本研究旨在了解响应T细胞活化而发生的CD45基因的调节剪接，作为表征信号诱导的选择性剪接机制的模型。先前的研究表明，rna结合蛋白PSF通过与受调节外显子上的剪接沉默复合物相关联，在激活时抑制CD45可变外显子的包含。为了确定细胞外刺激和序列背景如何影响CD45信号响应性沉默复合物的组装和功能，本提案的具体目的如下：(1)通过rna亲和纯化鉴定不同CD45可变外显子4和5上沉默复合物内的相关蛋白；(2)通过纯化静息细胞和活化细胞中功能不同的PSF相关复合物，并使用体外和基于细胞的测定来确认PSF相关蛋白的功能相关性，鉴定调节PSF募集到CD45沉默复合物的蛋白；(3)通过验证候选调节信号分子和分析PSF及相关蛋白的翻译后修饰，确定信号蛋白如何影响沉默者复合物的差异活性。总之，这些研究将全面了解细胞外刺激如何通过外显子沉默复合物改变PSF的功能，从而实现对CD45可变外显子的诱导抑制。因此，这些研究将为信号诱导剪接调控机制提供一个新的范式。此外，最小信号响应沉默子复合物的识别，以及对这种复合物如何在不同的外显子上形成的理解，将最终为预测可能受类似机制调节的外显子提供信息。许多人类细胞类型必须能够快速而精确地改变其蛋白质表达，以响应给定的刺激，以便适当地发挥作用并预防疾病状态。目前的研究表明，在细胞外刺激下调节蛋白表达和细胞功能的丰富机制是信号诱导的选择性剪接。本提案中的研究试图阐明细胞激活调节选择性剪接的一种机制，作为理解信号响应剪接调节的更广泛现象的模型。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how extracellular stimuli can induce a single gene to encode functionally distinct protein isoforms through the process of signal-induced alternative splicing. Alternative splicing is a major determinant of diversity within the human proteome and is a critical mechanism for modulating protein expression. An increasing number of genes have been shown to undergo alternative splicing in response to extracellular stimuli, particularly in the nervous and immune systems where cells must react robustly to changing environmental conditions. Moreover, mistakes in alternative splicing, including inhibition of signal-induced splicing regulation, have been linked to numerous human diseases. However, relatively little is understood about the proteins and mechanisms that regulate this process. This proposal seeks to understand the regulated splicing of the CD45 gene that occurs in response to T cell activation, as a model for characterizing mechanisms of signal-induced alternative splicing. Previous studies have demonstrated that the RNA-binding protein PSF represses inclusion the CD45 variable exons upon activation, by associating with a splicing silencer complex on the regulated exons. To determine how extracellular stimuli and sequence context influence the assembly and function of the CD45 signal-responsive silencer complex, the Specific Aims of this proposal are as follows: (1) To identify the proteins that associate within the silencer complex on the divergent CD45 variable exons 4 and 5 by RNA-affinity purification, (2) To identify proteins that regulate PSF recruitment to the CD45 silencer complexes, by purification of functionally distinct PSF-associated complexes from resting and activated cells and use of in vitro and cell-based assays to confirm the functional relevance of PSF associated proteins, and (3) To determine how signaling proteins influence the differential activity of the silencer complex by validation of candidate regulatory signaling molecules and analysis of post-translational modifications of PSF and associated proteins. Together these studies will provide a comprehensive understanding of how extracellular stimuli alters the function of PSF with exonic silencer complexes to achieve induced repression of the CD45 variable exons. As such, these studies will provide a new paradigm for mechanisms of signal-induced splicing regulation. In addition, the identification of a minimal signal-responsive silencer complex, and an understanding of how such a complex forms on distinct exons, will ultimately inform efforts to predict exons that may be regulated by similar mechanisms. Many human cell types 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 stimulation is that of signal-induced alternative splicing. The studies in this proposal seek to elucidate one mechanism by which cellular activation regulates alternative splicing, as a model for understanding the broader phenomena of signal-responsive splicing regulation.