RNA Binding Protein CUGBP2 in Intestinal Epithelium

描述（由申请人提供）：本申请的主要目标是了解RNA结合蛋白CUGBP2在肠上皮细胞mRNA稳定性和翻译的转录后水平调控基因表达的机制。这种蛋白在癌细胞中的过度表达会导致细胞死亡。我们已经确定CUGBP2与环氧化酶-2和Mcl-1 mrna的3'非翻译区富含au的序列相互作用，并在结合后下调这两种mrna的翻译。我们还确定CUGBP2水平在癌细胞中降低，但其表达在有丝分裂突变的细胞中升高。此外，CUGBP2在转录水平上受三种不同启动子的调控。位于常用的近端启动子上游的启动子的激活导致在蛋白质的n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的差异，但更重要的是蛋白质的活性。添加氨基酸的变异不影响细胞活力。此外，三种变异对靶基因剪接有不同的影响。基于这些观察，我们建议确定三个目标。在目标1中，我们将确定三个CUGBP2启动子受到调控的机制。此外，CUGBP2在选择性剪接的转录后水平上受到调控，导致在5'非翻译区包含一个内含子。我们将识别调节这一过程的顺式作用序列和反式作用因子。在Aim 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。我们将进行系统的删除和精细诱变，以确定CUGBP2中涉及RNA：蛋白质和蛋白质：蛋白质相互作用的结构域。此外，我们将确定在基础条件下和细胞暴露于3-辐照时调节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稳定性和翻译调控的影响。在Aim 3中，我们将确定CUGBP2基因是否是一种新的肿瘤抑制基因和原癌基因。我们将确定这三种亚型在人类结肠肿瘤组中的表达水平。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。这些实验的完成将有助于我们更好地了解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表达变化是否与肿瘤行为有关。公共卫生相关性：结直肠癌是美国癌症相关死亡的主要原因。了解正常细胞如何发展为癌症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蛋白质CUGBP2，它的表达在癌细胞中丢失。将蛋白质恢复到癌细胞中会杀死癌细胞。我们正在确定表达CUGBP2的基因是如何在癌细胞中被沉默的，这样我们就可以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.