Mechanisms of Signal-Induced Alternative Splicing: CD45

描述（由申请人提供）：本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，也是调节蛋白质表达的关键机制。越来越多的基因已被证明在响应细胞外刺激时进行选择性剪接，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烈反应。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，人们对调节这一过程的蛋白质和机制知之甚少。该提案旨在了解调节剪接的CD45基因，发生在响应T细胞活化，作为一个模型，表征信号诱导的选择性剪接的机制。先前的研究已经证明，RNA结合蛋白PSF通过与受调节的外显子上的剪接沉默子复合物相关联，在激活时抑制包含CD45可变外显子。为了确定细胞外刺激和序列环境如何影响CD45信号应答沉默物复合物的组装和功能，本提案的具体目的如下：（1）通过RNA亲和纯化鉴定在趋异的CD45可变外显子4和5上的沉默物复合物内缔合的蛋白质，（2）为了鉴定调节PSF募集至CD45沉默物复合物的蛋白质，通过从静息和活化细胞中纯化功能不同的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，（3）通过验证候选调节信号分子和分析PSF及其相关蛋白的翻译后修饰，确定信号蛋白如何影响沉默子复合物的差异活性。总之，这些研究将提供一个全面的了解如何细胞外刺激改变PSF与外显子沉默复合物的功能，以实现诱导的CD45可变外显子的抑制。因此，这些研究将为信号诱导的剪接调控机制提供新的范式。此外，最小的信号响应沉默复合物的鉴定，以及对这种复合物如何在不同外显子上形成的理解，将最终为预测可能受类似机制调控的外显子的努力提供信息。 许多人类细胞类型必须能够响应给定的刺激而快速精确地改变其蛋白质表达，以便适当地发挥功能并预防疾病状态。目前的研究表明，一个丰富的机制，调节蛋白质的表达和细胞功能的细胞外刺激是信号诱导的选择性剪接。本提案中的研究旨在阐明细胞活化调节选择性剪接的一种机制，作为理解信号响应性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.