RNA Binding Protein CUGBP2 in Intestinal Epithelium

肠上皮细胞中的 RNA 结合蛋白 CUGBP2

• 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
• 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' Untranslated Regions; 5' 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

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）：本申请的广泛目标是了解RNA结合蛋白CUGBP 2在肠上皮细胞中mRNA稳定性和翻译的转录后水平调节基因表达的机制。该蛋白在癌细胞中的过度表达导致细胞死亡。我们已经确定，CUGBP 2与环氧合酶-2和Mcl-1 mRNA的3 '非翻译区中富含AU的序列相互作用，并且在结合后下调两种mRNA的翻译。我们还确定了CUGBP 2水平在癌细胞中降低，但其表达在经历有丝分裂灾难的细胞中升高。此外，CUGBP 2在转录水平上由三种不同的启动子调控。来自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-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的差异，但更重要的是蛋白质的活性。具有添加的氨基酸的变体不影响细胞活力。此外，三种变体对靶基因剪接有不同的影响。根据这些观察，我们建议确定三个目标。在目标1中，我们将确定三个CUGBP 2启动子的调节机制。此外，CUGBP 2在转录后水平的选择性剪接中受到调节，导致在5 '非翻译区中新包含一个内含子。我们将确定顺式作用序列和反式作用因子调节这一过程。在目标2中，我们将确定CUGBP 2的细胞功能。我们已经鉴定了与CUGBP 2结合的细胞因子。我们将进行系统性缺失和精细诱变，以确定CUGBP 2中参与RNA：蛋白质和蛋白质：蛋白质相互作用的结构域。此外，我们将确定CUGBP 2结构域调节CUGBP 2定位在基础条件下，当细胞暴露于3-辐射。我们还将确定这些相互作用对CUGBP 2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CUGBP 2基因是否既是一种新的肿瘤抑制基因，也是一种原癌基因。我们将确定这三种亚型在一组人结肠肿瘤中的表达水平。此外，我们将确定不同的CUGBP 2亚型是否影响异种移植模型中的肿瘤行为。这些实验的完成应该让我们更好地了解RNA结合蛋白CUGBP 2在正常上皮细胞中的功能，以及在肿瘤细胞中观察到的CUGBP 2表达的变化是否是肿瘤行为的原因。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。我们已经鉴定了一种蛋白质，CUGBP 2，其在癌细胞中的表达丢失。将蛋白质恢复到癌细胞中会杀死细胞。我们正在确定表达CUGBP 2的基因如何在癌细胞中沉默，以便我们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。