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

上皮剪接调节蛋白的功能及其在 EMT 中的作用

• 批准号: 8086050
• 负责人: RUSS Paul CARSTENS
• 金额: $ 15.83万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-02 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8086050
• 关键词: Alternative Splicing; Binding; Binding Sites; Biological Assay; CD44 gene; Cell Line; Cells; Complementary DNA; Complex; Deletion Mutation; Development; Disease; Down-Regulation; Ectopic Expression; Ensure; Epithelial; Epithelial Cells; Event; Exons; FGFR2 gene; Gene Expression; Genes; Homeostasis; Luciferases; Maintenance; Malignant Neoplasms; Mediating; Mesenchymal; Modeling; Molecular; Mutation Analysis; Names; Neoplasm Metastasis; Organ; Paracrine Communication; Pathway interactions; Pattern; Phenotype; Process; Protein Binding Domain; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Interference; RNA Recognition Motif; RNA Splicing; Regulation; Role; Signal Pathway; Site; Tertiary Protein Structure; Transcript; Variant; base; cDNA Expression; cell growth; cell type; epithelial to mesenchymal transition; gain of function; genetic regulatory protein; genome-wide; high throughput screening; loss of function; novel; overexpression; paralogous gene; programs; protein protein interaction; restoration; small molecule; tumor

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).

FGFR 2外显子IIIb和IIIc的选择性剪接导致FGFR 2基因的细胞类型特异性表达。 上皮细胞中的FGFR 2-IIIb同种型和间充质细胞中的FGFR 2-IIIc，这种剪接选择是必需的 在发展过程中。我们先前假设上皮细胞类型特异性剪接程序， FGFR 2和其他转录物由上皮特异性剪接调节剂协调。在第一次成功使用 在全基因组范围内，高通量的cDNA表达筛选剪接因子，我们发现， 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相反。 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的调节因子 程序.我们将进一步描述ESRPs调节FGFR 2剪接的机制， 鉴定构成上皮特异性剪接的另外的共调节的可变剪接转录物 签名. ESRP 1和ESRP 2的表达在上皮间充质转化期间关闭 (EMT)因此，我们也将研究ESRP下调的作用和相应的变化， 在剪接共同调节的转录本，有助于EMT。 具体目标1.使用以下方法完成FGFR 2剪接调节子的鉴定： 建立了高通量、基于细胞的剪接测定。我们将完成全基因组的高通量 屏幕，以建立一套全面的拼接监管机构，与ESRP合作，在一般 上皮剪接程序。 具体目标2.研究ESRP 1和ESRP 2调节的机制 上皮细胞类型特异性剪接。ESRPs调节剪接的分子机制将 通过鉴定ESRP结合位点、必需蛋白结构域和功能来研究 相关的蛋白质相互作用。 具体目标3.识别上皮细胞类型特异性剪接标记， 受ESRP调控的可变剪接事件。使用剪接敏感 微阵列，我们将定义一个全面的一套生物连贯的转录本，共同调控的 除了FGFR 2、CD 44、ENAH和p120-Ctn之外的ESRP。 具体目标4.研究ESRPs在上皮间充质细胞中的作用 过渡（EMT）。我们将确定ESRPs表达的缺失是否是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）。