Deciphering the alternative splicing regulatory program driving the myofibroblast state

选择性剪接是一种强大的机制，通过允许来自单个基因的多个信使rna的转录来扩大蛋白质组的多样性。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）也有了很高的理解，最近最终形成了“剪接代码”。然而，对于控制剪接因子表达/活性的上游机制知之甚少，更不用说将细胞外因子与其他剪接调节联系起来了。我的实验室感兴趣的是破译一个替代剪接网络的上游调控机制，协调人类的关键发育程序。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，它们通过特征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的细胞外基质分泌能力。这可以通过体外用肿瘤生长因子刺激成纤维细胞来重现，肿瘤生长因子是肌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种选择性剪接异构体，包括已建立的纤维连接蛋白异构体，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，蛋白质组学方法鉴定了肌成纤维细胞中几种肿瘤生长因子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节控制（肿瘤生长因子β和剪接因子驱动肌成纤维细胞表型之间发生了什么）尚不清楚。因此，在这项NSERC - Discovery资助中，我假设在肿瘤生长因子β刺激下，控制一组剪接因子表达/活性的调节网络导致特定肌成纤维细胞剪接异构体的协调表达，这对肌成纤维细胞分化至关重要。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：总的来说，这项研究将产生一个完整的描述，即由一个替代剪接网络驱动的关键发育程序是如何被调节的，这将是我实验室的初级科学家获得尖端技术实践培训的一个独特机会。

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.