Mechanisms of hnRNPM in Alternative Splicing Regulation During EMT

描述（由申请人提供）：前体mRNA的选择性剪接是一种基本机制，通过该机制，基因可以产生多种不同的mRNA转录物，产生具有不同甚至相反功能的蛋白质同种型。尽管选择性剪接发生在几乎所有的人类基因中，但我们对调节选择性剪接的分子机制以及选择性剪接在正常和疾病状态中的作用的理解仍然是不完整和零散的。在这个项目中，我们建议调查的分子机制，选择性剪接是调节在发育过程中，称为上皮-间充质转化（EMT）。EMT涉及从紧密堆积的鹅卵石样上皮状态到能动的纺锤形间充质状态的变化。当异常激活时，EMT可导致包括肺、心脏和肾脏在内的许多组织纤维化，严重影响健康。异常激活的EMT还导致癌症转移，这是癌症相关死亡率的主要原因。不幸的是，驱动细胞进行EMT的确切机制尚未完全了解。通过对CD 44基因的研究，我们最近证明了选择性剪接调控因果控制EMT。CD 44编码通过选择性剪接产生的细胞表面蛋白家族。包含一个或多个可变外显子产生CD 44变体（CD 44 v），而跳过所有可变外显子产生CD 44标准（CD 44 s）。我们发现，CD 44选择性剪接在EMT过程中受到差异调节，导致上皮细胞中的CD 44 v表达转换为间充质细胞中的CD 44 s表达。重要的是，当CD 44亚型转换受到干扰时，细胞不再经历EMT。这些发现意味着，它是至关重要的，以确定调节可变剪接的机制，以更好地了解EMT和EMT相关疾病中的可变剪接的功能。为了实现这一目标，我们最近确定hnRNPM作为一个关键的剪接因子，刺激生产的CD 44，促进EMT表型。在拟议的研究中，我们将测试我们的假设，hnRNPM和其他剪接调节竞争或合作，以调节关键基因的选择性剪接，包括CD 44在EMT。为了验证这一假设，我们制定了以下具体目标：目标1。定义反式作用因子和顺式作用元件，在EMT过程中控制细胞类型依赖的选择性剪接。目标2.表征hnRNPM促进CD 44外显子跳跃的分子机制。目标3.定义hnRNPM调节的剪接事件，这对EMT至关重要。了解控制EMT的选择性剪接调节的分子机制对于开发干扰EMT再激活疾病（如组织纤维化和癌症转移）的治疗策略可能很重要。我们新定义的EMT关键可变剪接基因也可能为EMT相关人类疾病的发病机制和治疗提供新的思路。

DESCRIPTION (provided by applicant): Alternative splicing of pre-mRNA is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. Despite the fact that alternative splicing occur in nearly all human genes, our understanding of molecular mechanisms regulating alternative splicing and the role of alternative splicing in normal and disease states remains incomplete and fragmented. In this project we propose to investigate the molecular mechanisms by which alternative splicing is regulated during a developmental process, termed epithelial-mesenchymal transition (EMT). EMT involves the change from a tightly packed cobble-stone like epithelial state to a motile and spindle shaped mesenchymal state. When abnormally activated, EMT can cause fibrosis in many tissues including the lung, heart, and kidney, severely affecting health. Aberrantly activated EMT also results in cancer metastasis, the leading cause of cancer-related mortality. Unfortunately, the precise mechanism that drives cells to undergo EMT has not been fully understood. Through the study of the CD44 gene, we recently demonstrated that alternative splicing regulation causally controls EMT. CD44 encodes a family of cell surface proteins produced by alternative splicing. Inclusion of one or more of the variable exons generates CD44 variant (CD44v), whereas skipping all of the variable exons produces CD44 standard (CD44s). We discovered that CD44 alternative splicing is differentially regulated during EMT, resulting in a switch in expression from CD44v in epithelial cells to CD44s in mesenchymal cells. Importantly, when CD44 isoform switching is perturbed, cells can no longer undergo EMT. These findings imply that it is crucial to determine the mechanisms regulating alternative splicing in order to better understand the function of alternative splicing in EMT and EMT-associated diseases. Towards this goal, we have recently identified hnRNPM as a critical splicing factor that stimulates the production of CD44s, promoting an EMT phenotype. In the proposed studies, we will test our hypothesis that hnRNPM and other splicing regulators compete or cooperate to regulate alternative splicing of critical genes including CD44 during EMT. To test this hypothesis, we have developed the following Specific Aims: Aim 1. Define trans-acting factors and cis-acting elements that govern cell-type dependent alternative splicing during EMT. Aim 2. Characterize the molecular mechanism by which hnRNPM promotes CD44 exon skipping. Aim 3. Define hnRNPM-regulated splicing events that are critical for EMT. Understanding the molecular mechanisms of alternative splicing regulation that controls EMT could be important for the development of therapeutic strategies that perturb EMT-reactivated diseases such as tissue fibrosis and cancer metastasis. Our newly- defined alternatively spliced genes critical for EMT may also offer new ideas regarding the pathogenesis and treatment of EMT-associated human diseases.