RNA Binding Protein CUGBP2 in Intestinal Epithelium

描述(申请人提供)：本申请的主要目标是了解RNA结合蛋白CUGBP2在转录后水平上调节肠道上皮细胞中基因表达的机制，包括mRNA的稳定性和翻译。这种蛋白质在癌细胞中的过度表达会导致细胞死亡。我们已经确定CUGBP2与环氧合酶-2和Mcl-1mRNAs的3‘非翻译区富含AU的序列相互作用，并在结合时下调这两个mRNAs的翻译。我们还确定，在癌细胞中，CUGBP2水平降低，但在经历有丝分裂灾难的细胞中，其表达升高。此外，CUGBP2在转录水平上受三种不同启动子的调控。位于常用近端启动子上游的启动子的激活导致蛋白质的N-末端包含额外的氨基酸。我们的研究表明，这种添加的结果是蛋白质的细胞定位不同，但更重要的是蛋白质的活性不同。添加了氨基酸的变异体不会影响细胞活力。此外，三个变异体对靶基因剪接也有不同的影响。基于这些观察，我们提出了确定三个目标。在目标1中，我们将确定三个CUGBP2启动子的调控机制(S)。此外，CUGBP2在转录后水平上受到选择性剪接的调控，导致在5‘非翻译区新包含一个内含子。我们将确定调控这一过程的顺式作用序列和反式作用因子。在目标2中，我们将确定CUGBP2的细胞功能。我们已经确定了与CUGBP2结合的细胞因子。我们将进行系统的缺失和精细的突变，以确定CUGBP2中参与RNA：蛋白质和蛋白质：蛋白质相互作用的结构域。此外，我们还将确定在基础条件下和细胞受到3-射线照射时调节CUGBP2定位的CUGBP2结构域。我们还将确定这些相互作用对CUGBP2介导的RNA剪接、mRNA稳定性和翻译调节的影响。在目标3中，我们将确定CUGBP2基因是否既是一个新的抑癌基因，又是一个原癌基因。我们将确定这三种异构体在一组人类结肠肿瘤中的表达水平。此外，我们将确定不同的CUGBP2亚型是否会影响异种移植模型中的肿瘤行为。这些实验的完成应该会让我们更好地了解RNA结合蛋白CUGBP2在正常上皮细胞中的功能，以及在肿瘤细胞中观察到的CUGBP2表达的变化是否与肿瘤行为有关。公共卫生相关性：在美国，结直肠癌是癌症相关死亡的主要原因。了解正常细胞是如何发展成癌症的，将有助于我们开发针对这两种疾病的新疗法。我们已经确定了一种在癌细胞中缺失的蛋白质CUGBP2。将蛋白质恢复到癌细胞中会杀死这些细胞。我们正在确定表达CUGBP2的基因在癌细胞中是如何沉默的，以便我们能够确定逆转这一过程的方法。这可能会阻止或减缓肿瘤的发生。

DESCRIPTION (provided by applicant): The broad goal of this application is to understand the mechanisms by which RNA binding protein CUGBP2 regulates gene expression at the posttranscriptional level of mRNA stability and translation in intestinal epithelial cells. Overexpression of the protein in cancer cells results in cell death. We have determined that CUGBP2 interacts with AU-rich sequences in the 3'untranslated region of cyclooxygenase-2 and Mcl-1 mRNAs and upon binding downregulates the translation of both mRNAs. We have also determined that CUGBP2 levels are decreased in cancer cells but its expression is elevated in cells undergoing mitotic catastrophe. In addition, CUGBP2 is regulated at the levels of transcription by three different promoters. Activation from promoters located upstream from the commonly used proximal promoter results in the inclusion of additional amino acids in the N-terminus of the protein. Our studies suggest that the consequence of this addition is differences in cellular localization of the protein, but more importantly in the activity of the protein. The variants with the added amino acids do not affect cell viability. In addition, there are differential effects on target gene splicing with the three variants. Based on these observations, we have proposed to determine three aims. In Aim 1, we will determine the mechanism(s) by which the three CUGBP2 promoters are regulated. In addition, CUGBP2 is regulated at the posttranscriptional level of alternative splicing resulting in the novel inclusion of one intron in the 5'untranslated region. We will identify the cis-acting sequences and trans-acting factors regulating this process. In Aim 2, we will determine the cellular functions of CUGBP2. We have identified cellular factors that bind to CUGBP2. We will perform systematic deletion and fine mutagenesis to identify the domains in CUGBP2 that are involved in RNA:protein and protein:protein interactions. In addition, we will determine the CUGBP2 domains that regulate CUGBP2 localization under basal conditions and when the cells are exposed to 3-irradiation. We will also determine the effect of these interactions on CUGBP2 mediated RNA splicing, mRNA stability and translation regulation. In Aim 3, we will determine whether the CUGBP2 gene is both a novel tumor suppressor and a protooncogene. We will determine the expression levels of the three isoforms in a panel of human colon tumors. Furthermore, we will determine whether the different CUGBP2 isoforms affect tumor behavior in xenograft models. Completion of these experiments should give us a better understanding of how the RNA binding protein CUGBP2 functions in normal epithelial cells, and whether changes in the CUGBP2 expression that is observed in tumor cells is responsible for tumor behavior. PUBLIC HEALTH RELEVANCE: Colorectal cancer is the leading in cancer related deaths in the United States. Understanding how the normal cell progresses to a cancer will aid in our developing novel therapies for both diseases. We have identified a protein, CUGBP2 whose expression is lost in cancer cells. Restoring the protein into the cancer cell kills the cells. We are in the process of identifying how the gene expressing CUGBP2 is silenced in cancer cells so that we can determine ways to reverse the process. This might stop or slow down the tumorigenesis.