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，因为在这个过程中，主要信使(Pre-mRNA)通过包含和排除特定的蛋白质编码外显子集而变成多个成熟的信使。在广泛的生物过程中，细胞分化需要选择性剪接。尽管蛋白质组学很重要，但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。蛋白质组学是许多机械性生物学研究中研究全球蛋白质表达、修饰和相互作用的有力工具。到目前为止，蛋白质组学在研究选择性剪接方面的应用有限，这主要是由于缺乏适当的方法学。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控因子，对于进一步了解调控细胞分化的各种剪接机制是至关重要的。*我提出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，该框架机械地将信号转导、剪接因子和前mRNA底物联系起来，以控制细胞分化。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，因为它们可以在培养中方便地扩增，并能够分化为大多数下游细胞类型。*我们将在这项拟议的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确定关键选择性剪接事件的上游剪接调控因子，这些剪接事件在指导PSC命运控制方面至关重要。其次，我们将开发蛋白质组学方法来全面研究人类PSC分化启动前后的蛋白质剪接变体。最后，我们将利用生化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自我更新和分化启动中的功能。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知识。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。这个研究项目中开发的蛋白质组学方法可以应用于检测可能代表人类疾病(如癌症和神经退行性疾病)潜在治疗靶点的选择性剪接事件和上游调控因子。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，他们对加拿大由持续的科学和技术发展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.