Deciphering the alternative splicing regulatory program driving the myofibroblast state

选择性剪接是一种强大的机制，它通过允许从单个基因转录多个信使RNA来扩大蛋白质组的多样性。在文献中，许多例子都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(例如顺式中的RNA序列和反式中作用的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，目前对剪接因子表达/活性的上游调控机制知之甚少，更不用说将细胞外因子与选择性剪接调控联系起来。我的实验室对破译另一种剪接网络上游的调控机制感兴趣，该网络在人类体内编排了关键的发育程序。损伤后，成纤维细胞按照发育程序分化为肌成纤维细胞。通过这种方式，它们通过组装特有的应力纤维以及再生正常组织功能所需的额外细胞外基质分泌能力，获得增强的收缩性能。这可以通过在体外用肿瘤生长因子β刺激成纤维细胞来概括，肿瘤生长因子β是肌肉成纤维细胞分化的主要调节因子。我们发现了与肌成纤维细胞状态相关的多个选择性剪接亚型，包括已建立的纤维连接蛋白亚型，称为纤维连接蛋白额外结构域A，对肿瘤生长因子β诱导的肌成纤维细胞状态至关重要。此外，一种蛋白质组学方法确定了几种肿瘤生长因子β调节的肌成纤维细胞剪接因子。然而，这种选择性剪接程序的深度以及最终的上游调控(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)尚不清楚。因此，在这项NSERC-Discovery拨款中，我假设在肿瘤生长因子β刺激下，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异构体的协调表达。更确切地说，我们将在一名博士生和两名本科生的帮助下完成以下具体目标：目标1确定驱动肌成纤维细胞分化的替代剪接事件目的2为了全面确定驱动肌成纤维细胞分化的肌纤维母细胞剪接因子，这项研究将完整地描述由替代剪接网络驱动的关键发育计划是如何调控的，这将是我实验室的初级科学家获得前沿技术实践培训的独特机会。

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.