Mechanisms of hnRNPM in Alternative Splicing Regulation During EMT

hnRNPM 在 EMT 期间选择性剪接调控的机制

• 批准号: 8673352
• 负责人: Chonghui Cheng
• 金额: $ 29.36万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8673352
• 关键词: Activities of Daily Living; Affect; Alternative Splicing; Binding; Biological Assay; CD44 Antigens; CD44 gene; Calculi; Cancer Etiology; Cell Surface Proteins; Cells; Color; Complex; Development; Developmental Process; Disease; Elements; Engineering; Epithelial; Epithelial Cells; Event; Exons; Family; Fibrosis; Gene Expression Regulation; Gene Targeting; Genes; Goals; Health; Heart; Human; Indium; Introns; Kidney; Lung; Mammals; Mesenchymal; Messenger RNA; Molecular; Mus; Neoplasm Metastasis; Nuclear; Pathogenesis; Phenotype; Production; Protein Isoforms; Pyrimidine; RNA; RNA Splicing; Recruitment Activity; Regulation; Regulatory Element; Reporter; Reporting; Research; Ribonucleoproteins; Role; Shapes; Spliced Genes; Testing; Tissues; Trans-Activators; Transcript; Transfection; Variant; cell type; cis acting element; deep sequencing; exon skipping; genetic regulatory protein; human disease; mRNA Precursor; malignant breast neoplasm; mortality; novel; novel strategies; public health relevance; research study; therapeutic development; tumor; tumor progression

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.

描述（由申请人提供）：前MRNA的替代剪接是一种基本机制，通过该机制，基因可以产生多个不同的mRNA转录物，从而产生具有不同甚至相反的功能的蛋白质同工型。尽管几乎所有人类基因都出现了替代剪接，但我们对调节替代剪接的分子机制的理解以及替代剪接在正常和疾病状态中的作用仍然不完整和分散。在这个项目中，我们建议研究在发育过程中调节替代剪接的分子机制，称为上皮 - 间质转变（EMT）。 EMT涉及从类似上皮状态的紧密填充鹅卵石变为运动和主轴形间充质状态的变化。当异常激活时，EMT会在包括肺，心脏和肾脏在内的许多组织中引起纤维化，从而严重影响健康。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理解。通过对CD44基因的研究，我们最近证明了替代剪接调节因果控制EMT。 CD44编码通过替代剪接产生的细胞表面蛋白家族。包含一个或多个变量外显子会生成CD44变体（CD44V），而跳过所有可变外显子产生CD44标准（CD44S）。我们发现在EMT期间，CD44替代剪接受到差异调节，从而导致表达从上皮细胞中的CD44V转换为间质细胞中的CD44。重要的是，当CD44同工型切换受到干扰时，细胞将不再发生EMT。这些发现表明，确定调节替代剪接的机制至关重要，以便更好地了解EMT和EMT相关疾病中替代剪接的功能。为了实现这一目标，我们最近将HNRNPM确定为刺激CD44s产生的关键剪接因子，从而促进EMT表型。在拟议的研究中，我们将检验我们的假设，即HNRNPM和其他剪接调节器竞争或合作以调节关键基因的替代剪接，包括EMT期间CD44。为了检验这一假设，我们已经开发了以下特定目的：目标1。定义了控制EMT期间细胞类型依赖性替代剪接的跨作用因子和顺式作用元件。 AIM 2。表征HNRNPM促进CD44外显子跳过的分子机制。 AIM 3。定义对EMT至关重要的HNRNPM调节的剪接事件。了解控制EMT的替代剪接调节的分子机制对于开发治疗策略很重要，而治疗策略会扰乱EMT反应疾病，例如组织纤维化和癌症转移。我们新定义的对EMT至关重要的剪接基因也可能提供有关EMT相关人类疾病的发病机理和治疗的新想法。