POSTTRANSCRIPTIONAL GENE REGULATION IN T CELL ACTIVATION

T 细胞激活中的转录后基因调控

• 批准号: 6374361
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374361
• 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（根据调查员的摘要改编）：此应用程序是 致力于了解T细胞中细胞因子产生的机制 RNA结构和功能的水平。 T中免疫功能的调节 使Messenger RNA稳定性水平的细胞了解不足。我们将 通过生产来关注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，我们报道了伊拉夫蛋白与编码的mRNA结合并稳定 早期反应基因（ERG）类型的生长调节蛋白。 ERG mRNA 编码原子基因和细胞因子，它们独特地包含富集的不稳定性 Elav/Hu蛋白结合的3'未翻译区域中的元素。 因此，我们将使用使用 TET诱导系统并使用CD3/CD28途径研究其激活 其中的细胞因子mRNA先前已显示为稳定。在 此外，将使用体外降解/降解系统检查 HUR参与细胞因子mRNA的稳定性和 翻译。我们将使用HUR的站点定向的诱变来确定 t中细胞因子和ERG mRNA表达至关重要的相互作用 细胞激活。我们的假设是，HUR对于T细胞生长至关重要 通过管理细胞因子和ERG mRNA的表达来开发 与翻译设备的相互作用。因此，HUR上调 激活的T细胞将被预测允许这些细胞稳定细胞因子 mRNA并扩大免疫反应。此外，我们将在 T细胞在T细胞激活中的体内HUR靶标由 使用cDNA阵列多路复用。这种方法有可能识别 参与T细胞激活的新基因，这些基因在转录后是 受监管。总而言之，将检查HUR的RNA识别特异性和 互动，它对其对稳定性或翻译的影响 T细胞活化过程中的细胞因子和ERG mRNA。