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

FGFR2外显子IIIb和IIIc的选择性剪接导致细胞类型特异性的表达 FGFR2-IIIb在上皮细胞中的亚型和FGFR2-IIIc在间充质细胞中的异构体，这种剪接选择是必不可少的 在开发过程中。我们之前假设了一个上皮细胞类型的特异性剪接程序，在这个程序中 FGFR2和其他转录本由上皮特异的剪接调节因子协调。在第一次成功使用时 在全基因组、高通量的剪接因子cdna表达筛查中，我们发现了必不可少的 上皮特异的FGFR2剪接调控因子，命名为上皮剪接调控蛋白1和2 (ESRP1和ESRP2)。任一蛋白在间充质细胞中的异位表达将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个因子则相反 效果。CD44、ENAH和p120-CTN作为ESRP1和ESRP2剪接靶点的进一步鉴定 暗示这些蛋白质是更广泛的上皮转录后基因表达的调节者 程序。我们将进一步研究ESRP调控FGFR2和FGFR2剪接的机制。 确定构成上皮特异性剪接的其他共调控或剪接转录本 签名。ESRP1和ESRP2在上皮间质转化过程中的表达均被关闭 (EMT)，因此我们还将调查ESRP下调的作用和相应的变化 在共同调节的转录本的拼接中，这对EMT有贡献。 具体目标1.完成FGFR2剪接调节子的鉴定 建立了高通量、基于细胞的剪接分析。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节器，与一般的ESRP合作 上皮剪接程序。 特定目标2.研究ESRP1和ESRP2调节机制 上皮细胞特定类型的剪接。ESRPs调控剪接的分子机制将 通过鉴定ESRP结合部位、必需蛋白结构域和功能 相关的蛋白质-蛋白质相互作用。 特定目标3.鉴定上皮细胞类型特异的剪接信号 受ESRP调控的受监管的替代剪接事件。使用敏感的剪接 我们将定义一套全面的生物相关转录本，它们由 除FGFR2、CD44、ENAH和p120-CTN外，还有ESRPs。 特定目标4.研究ESRPs在上皮间充质中的作用 过渡(EMT)。我们将确定ESRPs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)。

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).