POSTTRANSCRIPTIONAL GENE REGULATION IN T CELL ACTIVATION

T 细胞激活中的转录后基因调控

• 批准号: 6604179
• 负责人: JACK D KEENE
• 金额: $ 30.8万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604179
• 关键词: T lymphocyte; cell growth regulation; chemical stability; cytokine; gene targeting; genetically modified animals; immunogenetics; intermolecular interaction; laboratory mouse; leukocyte activation /transformation; messenger RNA; nucleic acid structure; posttranscriptional RNA processing; regulatory gene

DESCRIPTION (adapted from investigator's abstract): This application is directed at understanding mechanisms of cytokine production in T cells at the level of RNA structure and function. The regulation of immune function in T cells at the level of messenger RNA stability is poorly understood. We will focus on the role of the ELAV/Hu proteins in T cells by production of HuR-deficient mice using the cre/lox mediated T cell knockout procedure. Previously, we reported that ELAV proteins bind to and stabilize mRNAs encoding growth regulatory proteins of the early-response gene (ERG) type. ERG mRNAs encode protooncogenes and cytokines, which uniquely contain AU-rich instability elements in their 3' untranslated regions to which the ELAV/Hu proteins bind. Therefore, we will overexpress and under-express HuR in Jurkat T cells using the Tet-inducible system and study their activation using the CD3/CD28 pathway in which cytokine mRNAs have been shown previously to be stabilized. In addition, an in vitro deadenylation/degradation system will be used to examine the mechanisms by which HuR engages cytokine mRNAs in stability and translation. We will use site-directed mutagenesis of HuR to determine interactions critical for the expression of cytokine and ERG mRNAs during T cell activation. Our hypothesis is that HuR is critical for T cell growth and development by governing the expression of cytokine and ERG mRNAs through interactions with the translational apparatus. Therefore, HuR upregulation in activated T cells would be predicted to allow these cells to stabilize cytokine mRNAs and to amplify the immune response. Additionally, we will identify in vivo HuR targets during T cell activation into distinct functional subsets by multiplexing using cDNA arrays. This approach has the potential to identify novel genes involved in T cells activation, which are posttranscriptionally regulated. In summary, HuR will be examined for RNA recognition specificity and interactions, which govern its effects on the stability or translation of cytokine and ERG mRNAs during T cell activation.

描述（改编自研究者摘要）：本申请是 旨在了解T细胞中细胞因子产生的机制， RNA的结构和功能。T细胞免疫功能的调节 细胞在信使RNA水平的稳定性知之甚少。我们将 关注ELAV/Hu蛋白在T细胞中的作用， 使用cre/lox介导的T细胞敲除程序的HuR缺陷小鼠。 以前，我们报道了ELAV蛋白结合并稳定编码ELAV的mRNA， 早期反应基因（ERG）型生长调节蛋白。ERG mRNA 编码原癌基因和细胞因子，其独特地含有富含AU的不稳定性 ELAV/Hu蛋白结合的3'非翻译区的元件。 因此，我们将在Jurkat T细胞中过表达和低表达HuR， Tet-inducible系统，并使用CD 3/CD 28途径研究其活化 其中细胞因子mRNA先前已被证明是稳定的。在 此外，将使用体外去腺苷化/降解系统来检查 HuR参与细胞因子mRNA稳定性的机制， 翻译.我们将使用HuR的定点突变来确定 T细胞中细胞因子和ERG mRNA表达的关键相互作用 细胞激活我们的假设是HuR对T细胞生长至关重要， 通过调控细胞因子和ERG mRNA的表达， 与平移装置的相互作用。因此，HuR上调， 预计活化的T细胞允许这些细胞稳定细胞因子， 并增强免疫反应。此外，我们将在 体内HuR在T细胞活化期间靶向不同的功能亚群， 使用cDNA阵列进行多路复用。这种方法有可能识别 新的基因参与T细胞活化，这是转录后 监管.总之，将检查HuR的RNA识别特异性， 相互作用，这决定了它对稳定性或翻译的影响。 细胞因子和ERG mRNA在T细胞活化过程中的作用。