Proteomic investigation of alternative splicing in the control of cell differentiation

选择性剪接在真核生物中产生蛋白质组多样性中起着重要作用，因为初级信使（前mRNA）在此过程中通过包含和排除特定的蛋白质编码外显子组而转变为多个成熟信使。选择性剪接是多种生物过程中细胞分化所必需的。尽管其重要性，在细胞分化过程中控制选择性剪接的精确机制仍然知之甚少。蛋白质组学是研究蛋白质表达、修饰和相互作用的有力工具。到目前为止，蛋白质组学研究选择性剪接的应用有限，主要是由于缺乏适当的方法。发展和应用蛋白质组学方法来研究细胞命运变化过程中的全局剪接程序和上游剪接调节因子，对于进一步理解调节细胞分化的选择性剪接机制至关重要。** 我的研究计划旨在开发和应用蛋白质组学方法来描绘一个总体框架，该框架将信号转导，剪接因子和前mRNA底物在控制细胞分化中联系起来。我们将使用人类多能干细胞（PSC）作为我们的细胞分化模型系统，因为它们可以方便地在培养中扩增，并且能够分化成大多数下游细胞类型。** 我们将在拟议的NSERC发现补助金计划中实现三个具体目标。首先，我们将开发蛋白质组学方法来鉴定在指导PSC命运控制中至关重要的关键选择性剪接事件的上游剪接调节子。第二，我们将开发蛋白质组学方法，以全面调查蛋白质剪接变异体在人类PSC分化开始之前和之后。最后，我们将使用生物化学和遗传学的方法来了解剪接变异体及其上游调控因子在人类PSC自我更新和分化起始中的功能。通过这些研究，我们将对真核细胞中的选择性剪接调控机制产生新的认识。这些结果也将提高我们对干细胞和祖细胞自我更新和分化机制的理解。在这项研究计划中开发的蛋白质组学方法可以应用于检测选择性剪接事件和上游调节因子，这些事件和上游调节因子可能代表人类疾病（如癌症和神经退行性疾病）中的潜在治疗靶点。此外，这项研究计划将使我的实验室能够培养有才华的年轻科学家，他们对加拿大持续的科学和技术发展所推动的可持续经济增长至关重要。

Alternative splicing plays an important role in generating proteome diversity in eukaryotes, as primary messengers (pre-mRNA) turn into multiple mature messengers by inclusion and exclusion of specific sets of protein-coding exons during the process. Alternative splicing is required for cell differentiation in a wide range of biological processes. Despite its importance, the precise mechanisms controlling alternative splicing during cell differentiation remain poorly understood.******Proteomics is a powerful tool to study global protein expression, modification, and interaction in many mechanistic biological studies. To date there has been limited use of proteomics to study alternative splicing, mainly due to the lack of proper methodology. Development and application of proteomic methods to study the global splicing program and the upstream splicing regulators during cell fate changes will be essential to further our understanding of the alternative splicing mechanisms that regulate cell differentiation. ******My proposed research program seeks to develop and apply proteomic methods to delineate a general framework that mechanistically links signal transduction, splicing factors, and pre-mRNA substrates in controlling cell differentiation. We will use human pluripotent stem cells (PSC) as our model systems of cell differentiation, as they can be conveniently expanded in culture and are able to differentiate into most downstream cell types. ******We will pursue three specific aims within this proposed NSERC Discovery Grant program. First, we will develop proteomic method to identify upstream splicing regulators of key alternative splicing events that are crucial in directing PSC fate control. Second, we will develop proteomic methods to globally investigate protein splice variants in human PSC before and after differentiation initiation. Last, we will use biochemical and genetic approaches to understand the functions of splice variants and their upstream regulators in human PSC self-renewal and differentiation initiation. Through these studies, we will generate new knowledge about the alternative splicing regulatory mechanisms in eukaryotic cells. These results will also improve our understanding of stem cell and progenitor cell self-renewal and differentiation mechanisms. The proteomic methods developed in this research program can be applied to detect alternative splicing events and upstream regulators that may represent potential therapeutic targets in human diseases such as cancer and neurodegenerative diseases. Furthermore, this research program will allow my laboratory to develop talented young scientists who are crucial for Canada's sustainable economic growth fueled by continuous science and technology development.