Deciphering the alternative splicing regulatory program driving the myofibroblast state

破译驱动肌成纤维细胞状态的选择性剪接调控程序

• 批准号: RGPIN-2021-04035
• 负责人: Brosseau, JeanPhilippe
• 金额: $ 2.19万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742669
• 关键词: Deciphering; alternative; splicing; regulatory; program

Alternative splicing is a powerful mechanism that expands the proteome diversity by allowing the transcription of multiple messenger RNAs from a single gene. Numerous examples have illustrated the functional impact of alternative splicing in the literature. There is also a high level of understanding of the major factors influencing a splicing decision (e.g. RNA sequence in cis and RNA binding factors acting in trans), recently culminating into a "splicing code". However, there is not much known about the upstream mechanisms controlling the expression/activity of splicing factors and even less linking extracellular factors to alternative splicing regulation. My laboratory is interested in deciphering the regulatory mechanisms upstream of an alternative splicing network orchestrating key developmental programs in humans. Following injury, fibroblasts follow a developmental program to differentiate into myofibroblasts. This way, they acquire enhanced contractile properties through the assembly of the characteristic stress fibers, and additional extracellular matrix secretion capacity necessary to regenerate normal tissue function. This can be recapitulated in vitro by stimulating fibroblasts with the tumor growth factor beta, the master regulator of myofibroblast differentiation. We discovered multiple alternatively spliced isoform associated to the myofibroblast state including the well-established isoform of fibronectin called fibronectin extra domain A critical to the tumor growth factor beta-induced myofibroblast state. In addition, a proteomic approach identified several tumor growth factor beta-regulated splicing factors in myofibroblasts. Still, the depth of this alternative splicing program and ultimately its upstream regulatory control (what's happening between the tumor growth factor beta and the splicing factors driving the myofibroblast phenotype) is unknown. In this NSERC - Discovery grant, I, therefore, hypothesize that upon tumor growth factor beta stimulation, a regulatory network controlling the expression/activity of a set of splicing factors leads to the coordinate expression of specific myofibroblast splicing isoforms that are critical for myofibroblast differentiation. More precisely, we will complete the following specific aims with the help of a Ph.D. student and two undergraduate students: AIM 1 To determine the alternative splicing events driving myofibroblast differentiation AIM 2 To determine the myofibroblast splicing factors driving myofibroblast differentiation Overall, this study will generate a complete portrait of how key developmental program driven by an alternative splicing network are regulated and will be a unique opportunity for junior scientists in my lab to get hands-on training on cutting-edge technologies.

选择性剪接是一种强大的机制，通过允许从单个基因转录多个信使RNA来扩展蛋白质组多样性。在文献中有许多例子说明了选择性剪接的功能影响。对影响剪接决定的主要因素（例如顺式RNA序列和反式RNA结合因子）也有很高的理解，最近最终形成了“剪接密码”。然而，有没有太多的上游机制控制剪接因子的表达/活性，甚至更少的连接细胞外因子的选择性剪接调节。我的实验室有兴趣破译的调节机制上游的选择性剪接网络编排在人类的关键发展计划。损伤后，成纤维细胞遵循发育程序分化成肌成纤维细胞。通过这种方式，它们通过特征性应力纤维的组装获得增强的收缩特性，以及再生正常组织功能所需的额外细胞外基质分泌能力。这可以通过用肿瘤生长因子β刺激成纤维细胞（肌成纤维细胞分化的主要调节因子）在体外重演。我们发现了与肌成纤维细胞状态相关的多种选择性剪接同种型，包括对肿瘤生长因子β诱导的肌成纤维细胞状态至关重要的纤连蛋白的公认同种型，称为纤连蛋白额外结构域A。此外，蛋白质组学方法鉴定了肌成纤维细胞中的几种肿瘤生长因子β调节的剪接因子。尽管如此，这种选择性剪接程序的深度及其最终的上游调控（在肿瘤生长因子β和驱动肌成纤维细胞表型的剪接因子之间发生了什么）仍然是未知的。因此，在NSERC -发现资助中，我假设在肿瘤生长因子β刺激后，控制一组剪接因子的表达/活性的调控网络导致对肌成纤维细胞分化至关重要的特定肌成纤维细胞剪接同种型的协调表达。更准确地说，我们将在博士的帮助下完成以下具体目标。学生和两名本科生：目的1确定驱动肌成纤维细胞分化的选择性剪接事件目的2确定驱动肌成纤维细胞分化的肌成纤维细胞剪接因子。这项研究将产生一个完整的画像，如何关键的发展程序驱动的选择性剪接网络的监管，并将是一个独特的机会，初级科学家在我的实验室，以获得动手培训切割-边缘技术。