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

描述（由申请人提供）：FGFR2外显子IIIb和IIIc的选择性剪接导致上皮细胞中FGFR2-IIIb亚型和间充质细胞中FGFR2-IIIc亚型的细胞类型特异性表达，这种剪接选择在发育过程中是必不可少的。我们之前假设了一个上皮细胞类型特异性剪接程序，其中FGFR2和其他转录本由上皮特异性剪接调节剂协调。在首次成功使用全基因组高通量cDNA表达筛选剪接因子中，我们发现了必不可少的上皮特异性FGFR2剪接调节因子，并将其命名为上皮剪接调节蛋白1和2 （ESRP1和ESRP2）。间充质细胞中任意一种蛋白的异位表达可将FGFR2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种因子则具有相反的效果。进一步鉴定CD44、ENAH和p120-ctn是ESRP1和ESRP2的剪接靶点，暗示这些蛋白是更广泛的上皮转录后基因表达程序的调节因子。我们将进一步表征esrp调控FGFR2剪接的机制，并鉴定构成上皮特异性剪接特征的其他共调控的可选剪接转录物。ESRP1和ESRP2的表达在上皮间充质转化（Epithelial Mesenchymal Transition， EMT）过程中被关闭，因此我们还将研究ESRP下调的作用，以及参与EMT的共调节转录物剪接的相应变化。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剪接调节因子的鉴定。我们将完成全基因组的高通量筛选，以建立一套全面的剪接调节因子，在一般的上皮剪接程序中与esrp合作。具体目标2。研究ESRP1和ESRP2调控上皮细胞类型特异性剪接的机制。ESRP调节剪接的分子机制将通过鉴定ESRP结合位点、必需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。具体目标3。鉴定一个上皮细胞类型特异性剪接信号，这些剪接事件是由esrp调控的。使用剪接敏感微阵列，我们将定义除FGFR2、CD44、ENAH和p120-Ctn外，由esrp共同调控的一套全面的生物学上一致的转录本。具体目标4。探讨esrp在上皮间质转化（EMT）中的作用。我们将确定EMT的发生是否需要ESRP表达的缺失，以及ESRP表达是否足以诱导间充质向上皮转化（MET）。公共卫生相关性：选择性剪接是一个非常重要的过程，在该过程中，同一基因可以产生具有不同功能的不同蛋白质，该过程的改变与包括癌症在内的许多疾病有关。我们已经确定了一种重要的选择性剪接调节蛋白，该蛋白在特定细胞类型上皮细胞中表达，并且是许多器官发育所必需的。该基因表达的缺失可能是导致许多癌症侵袭和转移的一个过程。

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). PUBLIC HEALTH RELEVANCE: Alternative splicing is a fundamentally important process in which the same gene can generate different proteins that have different functions, and alterations in this process are implicated in numerous diseases, including cancer. We have identified an essential alternative splicing regulatory protein that is expressed in a specific cell type, the epithelial cell, and which is required for development of many organs. The loss of expression of this gene may be one process that contributes to the invasion and metastasis of many cancers.