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.
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