Mechanisms of hnRNPM in Alternative Splicing Regulation During EMT

描述（由申请人提供）：前mRNA的选择性剪接是基因产生多个不同mRNA转录本的基本机制，产生具有不同甚至相反功能的蛋白质异构体。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不完整和碎片化。在这个项目中，我们建议研究在发育过程中被称为上皮-间质转化（EMT）的选择性剪接调节的分子机制。EMT包括从紧密堆积的鹅卵石样上皮状态到可运动的纺锤形间充质状态的变化。当异常激活时，EMT会导致包括肺、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也会导致癌症转移，这是癌症相关死亡的主要原因。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CD44基因的研究，我们最近证明了选择性剪接调控可以控制EMT。CD44编码一个由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CD44变体（CD44v），而跳过所有可变外显子产生CD44标准（CD44s）。我们发现CD44选择性剪接在EMT过程中受到差异调节，导致上皮细胞中的CD44v表达转换为间充质细胞中的CD44s表达。重要的是，当CD44异构体转换受到干扰时，细胞不能再进行EMT。这些发现表明，为了更好地了解选择性剪接在EMT和EMT相关疾病中的功能，确定调节选择性剪接的机制至关重要。为了实现这一目标，我们最近发现hnRNPM是刺激cd44产生的关键剪接因子，促进EMT表型。在拟议的研究中，我们将验证我们的假设，即hnRNPM和其他剪接调节剂在EMT期间竞争或合作调节包括CD44在内的关键基因的选择性剪接。为了验证这一假设，我们制定了以下具体目标：目标1。定义在EMT过程中控制细胞类型依赖性选择性剪接的反式作用因子和顺式作用因子。目标2。表征hnRNPM促进CD44外显子跳变的分子机制。目标3。定义对EMT至关重要的hnrnpm调节的剪接事件。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）的治疗策略非常重要。我们新定义的对EMT至关重要的选择性剪接基因也可能为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.