Regulation of alternative splicing during epithelial-mesenchymal transition

摘要 前-mRNA的选择性剪接使一个基因能够产生多个不同的mRNA转录本， 产生具有不同甚至相反功能的蛋白质亚型。我们研究的长期目标是 了解选择性剪接对生物活性的分子机制和功能。的关注点 本研究旨在探讨选择性剪接在上皮-间充质转化(EMT)中的调控作用。 EMT是一个重要的发育过程，它允许细胞从紧密堆积的鹅卵石般的 上皮细胞状态转变为可运动的梭形间充质细胞状态。当异常激活时， EMT促进许多类型的疾病，包括组织纤维化和癌症转移。通过在 RNA剪接和细胞生物学的交叉，我的实验室已经取得了几个重要的发现，将RNA 监管和EMT。我们的工作首次揭示了CD44基因剪接异构体切换的因果关系 控制EMT。通过操纵CD44选择性剪接，我们能够将上皮细胞之间的细胞 和间充质状态。我们还发现RNA结合蛋白(RBP)hnRNPM是一种关键的剪接 通过调节选择性剪接促进EMT的因子。这些结果表明，RNA剪接 调节可能是提供细胞可塑性的重要机制。通过改变 通过选择性剪接，细胞能够在上皮细胞状态和间充质细胞状态之间转换。这 逆转表型的能力对于正常发育的EMT以及癌症转移都是重要的。 要充分理解这一现象及其潜在的机制，需要有重大的新发现。我们的 建议的研究计划集中在(1)确定剪接异构体转换是否作为一种流行的 驱动EMT的机制，或者，如果它主要是一种副产品现象；(2)了解限制性商业惯例如何 精确控制EMT中的选择性剪接；以及(3)剖析信号级联如何向限制性商业惯例发出信号 并在EMT期间触发可选剪接改变。在过去的几年里，我们做出了重大努力 建立实验系统并获得专业知识，以帮助开展我们拟议的研究。这些努力包括 多种EMT系统中选择性剪接的大规模RNA图谱、生物信息学和实验 限制性商业惯例的分析，以及我们最近完成的EMT相关选择性剪接的激酶筛查 改装。完成拟议的工作将为我们对监管的理解提供新的见解 选择性剪接机制，从而有助于正常发育和疾病的生物学相关性。

ABSTRACT Alternative splicing of pre-mRNA enables a gene to give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. The long-term goal of our research is to understand the molecular mechanisms and function of alternative splicing on biological activities. The focus of this proposal is to investigate the regulation of alternative splicing in epithelial-mesenchymal transition (EMT). EMT is an essential developmental process that allows cells to change from a tightly packed cobble-stone-like epithelial cellular state to a motile and spindle-shaped mesenchymal cellular state. When abnormally activated, EMT promotes many types of diseases, including tissue fibrosis and cancer metastasis. Through working at the intersection of RNA splicing and cell biology, my lab has made several important findings connecting RNA regulation and EMT. Our work revealed, for the first time, that splice isoform switching of the CD44 gene causally controls EMT. By manipulating CD44 alternative splicing, we were able to convert cells between the epithelial and mesenchymal states. We have also identified the RNA-binding protein (RBP) hnRNPM as a critical splicing factor that promotes EMT through the regulation of alternative splicing. These results suggest that RNA splicing regulation could serve as an important mechanism that provides cellular plasticity. By shifting the programs of alternative splicing, cells are able to convert between the epithelial and mesenchymal cellular states. This capacity of reversing phenotypes is important for normal developmental EMT, as well as for cancer metastasis. Major new discoveries are necessary to fully understand this phenomenon and its underlying mechanisms. Our proposed research program is focused on (1) determining whether splice isoform switching acts as a prevalent mechanism that drives EMT or, if it is largely a phenomenon of byproducts; (2) understanding how RBPs precisely control alternative splicing during EMT; and (3) dissecting how signaling cascades elicit signals to RBPs and trigger alternative splicing changes during EMT. We have made major efforts in the past several years to build up experimental systems and gain expertise to help carry out our proposed studies. These efforts include large-scale RNA profiling of alternative splicing in multiple EMT systems, bioinformatics and experimental analysis of RBPs, as well as our recently completed kinase screen for EMT-associated alternative splicing alterations. Accomplishing the proposed work will provide new insights into our understanding of the regulatory mechanisms of alternative splicing, thus contributing to biological relevance in normal development and diseases.