POSTTRANSCRIPTIONAL GENE REGULATION IN T CELL ACTIVATION

描述(改编自调查员摘要)：本申请是 旨在了解T细胞中细胞因子的产生机制 RNA的结构和功能水平。T细胞免疫功能的调节 细胞在信使RNA水平上的稳定性还知之甚少。我们会 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蛋白与编码mRNAs结合并使其稳定 早期反应基因(ERG)类型的生长调节蛋白。ERG mRNAs 编码原癌基因和细胞因子，它们独特地包含富含AU的不稳定性 与ELAV/Hu蛋白结合的3‘非翻译区的元件。 因此，我们将在Jurkat T细胞中高表达和低表达Hur，使用 Tet诱导系统及其激活的CD3/CD28途径研究 其中细胞因子mRNAs先前已被证明是稳定的。在……里面 此外，将使用体外去烯化/降解系统来检查 HUR参与细胞因子mRNAs稳定和稳定的机制 翻译。我们将使用HUR的定点突变来确定 T细胞因子和ERG mRNAs表达的相互作用 细胞激活。我们的假设是，HUR对T细胞的生长和 调控细胞因子和ERG mRNAs表达的研究进展 与平移设备的相互作用。因此，HUR在 据预测，激活的T细胞可以使这些细胞稳定细胞因子 并放大免疫反应。此外，我们还将在 T细胞活化过程中的活体HUR靶点通过 使用cdna阵列进行多路传输。这种方法有可能识别出 参与T细胞激活的新基因，这些基因是转录后 受监管的。总之，将检查HUR的RNA识别特异性和 相互作用，控制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 mRNAs。

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.