Functions of Epithelial Splicing Regulatory Proteins and their role in the EMT

描述(申请人提供)：FGFR2外显子IIIb和IIIc的选择性剪接导致FGFR2-IIIb亚型在上皮细胞中的表达，以及FGFR2-IIIc在间充质细胞中的表达，这种剪接选择在发育过程中是必不可少的。我们先前假设了一个上皮细胞类型的特异性剪接程序，其中FGFR2和其他转录本由上皮特异性剪接调节因子协调。在第一次成功地使用全基因组、高通量的cDNA表达筛查剪接因子时，我们发现了必需的上皮特异的FGFR2剪接调节因子，并将其命名为上皮剪接调控蛋白1和2(ESRP1和ESRP2)。在间充质细胞中异位表达任何一种蛋白都会将FGFR2的剪接转换为上皮模式，而通过RNA干扰在上皮细胞中耗尽这两个因子则具有相反的效果。进一步鉴定CD44、ENAH和p120-CTN为ESRP1和ESRP2的剪接靶标，暗示这些蛋白是更广泛的上皮转录后基因表达计划的调节因子。我们将进一步研究ESRP调控FGFR2剪接的机制，并识别构成上皮特异性剪接信号的额外共调控或剪接转录本。在上皮间充质转化(EMT)过程中，ESRP1和ESRP2的表达都被关闭，因此我们还将研究ESRP下调的作用以及参与EMT的共同调控转录的剪接的相应变化。具体目标1：使用已建立的高通量、基于细胞的剪接分析完成FGFR2剪接调节子的鉴定。我们将完成全基因组、高通量的筛选，以建立一套全面的剪接调控因子，在一般的上皮剪接计划中与ESRP合作。研究ESRP1和ESRP2调控上皮细胞类型特异性剪接的机制。ESRP调控剪接的分子机制将通过鉴定ESRP结合位点、必要的蛋白质结构域和功能相关的蛋白质-蛋白质相互作用来研究。特定目标3.确定受ESRPs调控的共同调节的选择性剪接事件的上皮细胞类型特异性剪接特征。使用剪接敏感的微阵列，我们将定义一套全面的生物一致性转录本，这些转录本除了FGFR2、CD44、ENAH和p120-CTN外，还受到ESRPs的共同调控。具体目标4：研究ESRPs在上皮间质转化(EMT)中的作用。我们将确定ESRP的表达缺失是否是发生EMT所必需的，以及ESRP的表达是否足以诱导间充质向上皮的转变(MET)。

DESCRIPTION (provided by applicant): Alternative splicing of FGFR2 exons IIIb and IIIc results in the cell type-specific expression of the FGFR2-IIIb isoform in epithelial cells and FGFR2-IIIc in mesenchymal cells and this splicing choice is essential during development. We previously hypothesized an epithelial cell-type specific splicing program in which FGFR2 and other transcripts is coordinated by epithelial-specific splicing regulators. In the first successful use of a genome-wide, high throughput cDNA expression screen for splicing factors, we discovered essential epithelial-specific FGFR2 splicing regulators and named them Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2). Ectopic expression of either protein in mesenchymal cells switches FGFR2 splicing to the epithelial pattern, whereas depletion of both factors in epithelial cells via RNA interference has the opposite effect. Further identification of CD44, ENAH, and p120-ctn as splicing targets of ESRP1 and ESRP2 implicates these proteins as the regulators of a broader epithelial post-transcriptional gene expression program. We will further characterize the mechanisms by which the ESRPs regulate splicing of FGFR2 and identify additional co-regulated alternatively spliced transcripts that constitute an epithelial-specific splicing signature. Expression of both ESRP1 and ESRP2 is turned off during the Epithelial Mesenchymal Transition (EMT) and therefore we will also investigate the role of ESRP downregulation and the corresponding changes in splicing of co-regulated transcripts that contribute to the EMT. Specific Aim Number 1. Complete the identification of FGFR2 splicing regulators using established high throughput, cell-based splicing assays. We will complete genome-wide, high-throughput screens to establish a comprehensive set of splicing regulators that cooperate with the ESRPs in the general epithelial splicing program. Specific Aim Number 2. Investigate the mechanisms by which ESRP1 and ESRP2 regulate epithelial cell type-specific splicing. The molecular mechanisms by which the ESRPs regulate splicing will be investigated through the identification of ESRP binding sites, essential protein domains, and functionally relevant protein-protein interactions. Specific Aim Number 3. Identify an epithelial cell type-specific splicing signature of co- regulated alternative splicing events that are regulated by the ESRPs. Using splicing sensitive microarrays we will define a comprehensive set of biologically coherent transcripts that are co-regulated by the ESRPs in addition to FGFR2, CD44, ENAH, and p120-Ctn. Specific Aim Number 4. Investigate the role of the ESRPs in the Epithelial Mesenchymal Transition (EMT). We will determine whether the loss of ESRPs expression is required for the EMT to occur and whether ESRP expression sufficient to induce a Mesenchymal to Epithelial Transition (MET).