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

描述（由申请方提供）：FGFR 2外显子IIIb和IIIc的选择性剪接导致上皮细胞中FGFR 2-IIIb同种型和间充质细胞中FGFR 2-IIIc的细胞类型特异性表达，这种剪接选择在发育过程中至关重要。我们先前假设了一种上皮细胞类型特异性剪接程序，其中FGFR 2和其他转录物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，我们发现了必需的上皮特异性FGFR 2剪接调节因子，并将其命名为上皮剪接调节蛋白1和2（ESRP 1和ESRP 2）。间充质细胞中任一蛋白质的异位表达将FGFR 2剪接切换到上皮模式，而通过RNA干扰在上皮细胞中耗尽这两种因子具有相反的效果。进一步鉴定CD 44、ENAH和p120-ctn作为ESRP 1和ESRP 2的剪接靶点，暗示这些蛋白质作为更广泛的上皮转录后基因表达程序的调节剂。我们将进一步表征ESRPs调节FGFR 2剪接的机制，并鉴定构成上皮特异性剪接特征的其他共调节可变剪接转录物。ESRP 1和ESRP 2的表达在上皮间充质转化（EMT）期间关闭，因此我们还将研究ESRP下调的作用以及有助于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、基于细胞的剪接测定法完成FGFR 2剪接调节因子的鉴定。我们将完成全基因组范围的高通量筛选，以建立一套全面的剪接调节剂，与ESRPs在一般上皮剪接程序。具体目标2.研究ESRP 1和ESRP 2调节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、必需蛋白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.鉴定由ESRP调节的共调节的可变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，我们将定义一个全面的生物连贯的转录本，除了FGFR 2，CD 44，ENAH，和p120-Ctn的ESRPs的共同调节。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表达的丧失是否是EMT发生所必需的，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，在这个过程中，相同的基因可以产生具有不同功能的不同蛋白质，这个过程中的改变与许多疾病有关，包括癌症。我们已经确定了一个重要的选择性剪接调控蛋白，表达在一个特定的细胞类型，上皮细胞，这是许多器官的发展所必需的。该基因表达的缺失可能是导致许多癌症的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.