RNA Binding Protein CUGBP2 in Intestinal Epithelium

肠上皮细胞中的 RNA 结合蛋白 CUGBP2

基本信息

批准号：
7583130
负责人：
Shrikant Anant
金额：
$ 35.53万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7583130
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Adenocarcinoma Adenoviruses Affect Alternative Splicing Amino Acids Apoptosis Apoptotic Area Behavior Binding Cell Death Cell Survival Cell physiology Cells Cessation of life Cis-Acting Sequence Classification Code Colon Colon Carcinoma Colonic Neoplasms Colorectal Cancer Complementary DNA Diagnosis Disease Epithelial Cells Event Exclusion Exons Family Gene Expression Gene Targeting Genes Genetic Transcription Goals Human Intestines Introns Luciferases Malignant Neoplasms Mediating Messenger RNA Microtubule-Associated Proteins Mitotic Mus Mutagenesis Neoplasm Metastasis Normal Cell Nuclear Nuclear Export Nude Mice Open Reading Frames Pathway interactions Physiological Process Protein Isoforms Protein Overexpression Proteins Proto-Oncogenes RNA Binding RNA Splicing RNA-Binding Proteins Recurrence Regulation Research Role Signal Transduction Spliced Genes Therapeutic Therapeutic Intervention Trans-Activators Translations Tumor Promoters Tumor Suppressor Genes Tumor Suppressor Proteins United States Variant Vascularization Xenograft Model adenoma base cancer cell cell killing cis acting element cyclooxygenase 2 hnRNP A1 in vivo intestinal epithelium irradiation mRNA Stability member neoplastic cell novel overexpression promoter protein protein interaction public health relevance research study response tumor tumor growth tumor xenograft tumorigenesis

项目摘要

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.

描述（由申请人提供）：此应用的广泛目标是了解RNA结合蛋白CUGBP2在mRNA稳定性的转录后水平和肠上皮细胞中翻译的基因表达的机制。癌细胞中蛋白质的过表达导致细胞死亡。我们已经确定CUGBP2与环氧酶-2和Mcl-1 mRNA的3'untranslated区域中的富含AU序列相互作用，并在结合后下调两个mRNA的翻译。我们还确定，癌细胞中CUGBP2水平降低，但其表达在发生有丝分裂灾难的细胞中升高。此外，CUGBP2在转录水平受到三个不同启动子的调节水平。来自通常使用的近端启动子上游的启动子的激活导致在蛋白质的N末端中包含其他氨基酸。我们的研究表明，这种添加的结果是蛋白质的细胞定位差异，但更重要的是蛋白质的活性。带有添加氨基酸的变体不会影响细胞活力。另外，对靶基因剪接具有不同的影响，并具有三种变体的靶基因剪接。基于这些观察结果，我们建议确定三个目标。在AIM 1中，我们将确定对三个CUGBP2启动子受到调节的机制。此外，在替代剪接后的转录后水平上调节CUGBP2，导致新的包含在5'untranslated区域中的一个内含子。我们将确定调节此过程的顺式作用序列和跨作用因素。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。我们将执行系统的缺失和精细诱变，以识别RNA中涉及的CUGBP2中的结构域：蛋白质和蛋白质：蛋白质相互作用。此外，我们还将确定在基础条件下以及细胞暴露于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稳定性和翻译调节的影响。在AIM 3中，我们将确定CUGBP2基因是否既是一种新型的肿瘤抑制剂，又是原子能。我们将确定人类结肠肿瘤小组中三种同工型的表达水平。此外，我们将确定不同CUGBP2同工型是否影响异种移植模型中的肿瘤行为。这些实验的完成应使我们更好地了解正常上皮细胞中RNA结合蛋白CUGBP2的功能，以及在肿瘤细胞中观察到的CUGBP2表达的变化是否导致肿瘤行为。公共卫生相关性：大肠癌是美国与癌症相关的死亡的主要人物。了解正常细胞如何发展为癌症将有助于我们开发的两种疾病的新型疗法。我们已经鉴定出一种蛋白质CUGBP2，其表达在癌细胞中丧失。将蛋白质恢复到癌细胞中会杀死细胞。我们正在确定表达CUGBP2基因在癌细胞中如何沉默的过程，以便我们可以确定逆转过程的方法。这可能会停止或减慢肿瘤发生。