Proteomic investigation of alternative splicing in the control of cell differentiation

选择性剪接在真核生物蛋白质组多样性的产生中起着重要作用，在这一过程中，初级信使（pre-mRNA）通过包含和排除特定的蛋白质编码外显子转变为多个成熟信使。在广泛的生物过程中，细胞分化需要选择性剪接。尽管它很重要，但在细胞分化过程中控制选择性剪接的确切机制仍然知之甚少。******蛋白质组学是研究许多机制生物学研究中全局蛋白表达、修饰和相互作用的有力工具。迄今为止，由于缺乏适当的方法，蛋白质组学在研究选择性剪接方面的应用有限。开发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，对于进一步了解调节细胞分化的剪接机制至关重要。******我提出的研究计划旨在开发和应用蛋白质组学方法来描述一个总体框架，该框架将信号转导、剪接因子和mrna前底物在控制细胞分化中的机制联系起来。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。******我们将在这个提议的NSERC发现资助计划中追求三个具体目标。首先，我们将开发蛋白质组学方法来识别在指导PSC命运控制中至关重要的关键替代剪接事件的上游剪接调节因子。其次，我们将开发蛋白质组学方法来全面研究人类PSC分化开始前后的蛋白质剪接变异。最后，我们将利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认识。本研究项目中开发的蛋白质组学方法可用于检测替代剪接事件和上游调节因子，这些调节因子可能代表癌症和神经退行性疾病等人类疾病的潜在治疗靶点。此外，这个研究项目将使我的实验室能够培养有才华的年轻科学家，他们对加拿大在不断的科学和技术发展的推动下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.