ESRP regulated alternative splicing in EMT/MET and kidney development

ESRP 调节 EMT/MET 和肾脏发育中的选择性剪接

• 批准号: 8765611
• 负责人: Thomas W Bebee
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765611
• 关键词: Address; Alternative Splicing; Biological Models; Cell Communication; Cell Differentiation process; Cell Polarity; Cell physiology; Cells; Cytoskeleton; Development; Disease; Duct (organ) structure; Ductal Epithelium; Epithelial; Epithelial Cells; Epithelium; Event; Exons; Genes; Genetic Transcription; Health; Human; Individual; Kidney; Kidney Glomerulus; Knock-out; Laboratories; Maintenance; Mediating; Mesenchyme; Metanephric Diverticulum; Methodology; Mus; Pathway interactions; Pattern; Phenotype; Polyadenylation; Population; Process; Proteins; Proteome; Proteomics; RNA Sequences; RNA Splicing; Regulation; Renal tubule structure; Role; Spliced Genes; Structure; System; Testing; Tissue-Specific Splicing; Transcript; Tubular formation; Work; cell growth; cell type; epithelial to mesenchymal transition; genetic regulatory protein; in vivo; insight; migration; nephrogenesis; post-doctoral training; programs; protein function; research study

DESCRIPTION (provided by applicant): An important mechanism to increase proteome diversity is through the process of alternative splicing (AS), wherein individual gene transcripts can be processed to generate functionally distinct proteins, a phenomenon identified in nearly all multi-exon human genes. While most known splicing regulatory proteins are ubiquitously expressed, a subset of tissue specific splicing regulators that govern global splicing regulatory networks (SRN) have been identified. Our laboratory has identified the cell type-specific epithelial splicing regulatory proteins (ESRP1 and ESRP2) that regulate splicing of gene transcripts encoding proteins involved in the maintenance of epithelial cell differentiation and function. The process of epithelial to mesenchymal transition (EMT) and the reverse process of MET have been extensively studied at the transcriptional level, while only recently has the impact of alternative splicing during these processes been evaluated. The ESRPs are primary regulators of AS during the EMT, where a dramatic reduction in ESRP1 and ESRP2 is observed compared to only modest changes in expression of other splicing regulators. Notably, genes regulated at the level of transcription and A encode proteins that function in common pathways that impact EMT, but the specific genes altered at the transcriptional and AS levels do not overlap, emphasizing the importance of alternative splicing as an additional layer of regulation involved in cellular reprogrammin during EMT. Epithelial cell differentiation and MET conversions are functionally conserved mechanisms important for normal development and can be clearly observed in the formation of the kidney, where the ESRPs are expressed. Identification of the SRN mediated by ESRP will be crucial in understanding the mechanism of EMT/MET conversions and lend insight into these processes during normal development and disease. We hypothesize that ESRP driven alternative splicing events are essential in the proper regulation of epithelial cell differentiation and function, as well as the process of MET in normal kidney development and patterning. To test the role of the Esrps in kidney development we will utilize Cre-mediated conditional knockouts for the Esrps to evaluate: 1) Esrp function and splicing regulation during epithelial cell differentiation/specification in ureteric bud branching and formation of collecting duct epithelial cells and 2) Esrp function and splicing regulaton during MET in the formation of renal tubular cells from the cap mesenchyme. To address the impact of Esrp loss in kidney development and regulation of a SRN in vivo we will analyze kidneys at the histological level and at the level of alternative splicing and polyadenylation using state-of-the-art high throughput RNA sequencing methodologies.

描述(申请人提供)：增加蛋白质组多样性的一个重要机制是通过选择性剪接(AS)过程，其中单个基因转录本可以被处理以产生功能不同的蛋白质，这一现象几乎在所有多外显子人类基因中都存在。虽然大多数已知的剪接调控蛋白都是普遍表达的，但已经确定了管理全球剪接调控网络(SRN)的组织特异性剪接调控因子的子集。我们的实验室已经鉴定出 细胞类型特异的上皮剪接调节蛋白(ESRP1和ESRP2)，调节基因转录本的剪接，编码参与维持上皮细胞分化和功能的蛋白质。上皮向间充质转化(EMT)的过程和MET的逆转过程已经在转录水平上得到了广泛的研究，而直到最近才对这些过程中选择性剪接的影响进行了评估。ESRP是EMT期间AS的主要调节因子，与其他剪接调节因子的表达仅有轻微变化相比，ESRP1和ESRP2的表达显著减少。值得注意的是，在转录水平和A水平调节的基因编码的蛋白质在影响EMT的共同途径中发挥功能，但在转录水平和AS水平改变的特定基因并不重叠，强调了选择性剪接作为参与EMT过程中细胞重新编程的额外调节层的重要性。上皮细胞分化和MET转化是功能保守的机制，对正常发育很重要，在肾脏的形成过程中可以清楚地观察到，ESRPs在肾脏的形成中表达。识别ESRP介导的SRN对于理解EMT/MET转换的机制以及了解这些过程在正常发育和疾病中的作用至关重要。我们推测，ESRP驱动的选择性剪接事件在适当调节上皮细胞分化和功能以及MET在正常肾脏发育和模式中的过程中是必不可少的。为了测试ESRP在肾脏发育中的作用，我们将利用Cre介导的条件性基因敲除ESRP来评估：1)ESRP在输尿管芽分枝和集合管上皮细胞形成过程中上皮细胞分化/规范过程中的功能和剪接调控；2)ESRP功能和剪接调控在MET形成肾小管细胞过程中的作用。为了解决ESRP缺失对肾脏发育和体内SRN调控的影响，我们将使用最先进的高通量RNA测序方法，在组织水平和选择性剪接和多聚腺苷水平上分析肾脏。