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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI046451-03
Application #
6510938
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Nabavi, Nasrin N
Project Start
2000-07-01
Project End
2005-05-31
Budget Start
2002-07-01
Budget End
2003-05-31
Support Year
3
Fiscal Year
2002
Total Cost
$292,600
Indirect Cost
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Isoe, Jun; Stover, Weston; Miesfeld, R Barrett et al. (2013) COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities. Insect Biochem Mol Biol 43:732-9
Mack, Daniel J; Isoe, Jun; Miesfeld, Roger L et al. (2012) Distinct biological effects of golgicide a derivatives on larval and adult mosquitoes. Bioorg Med Chem Lett 22:5177-81
Alabaster, Amy; Isoe, Jun; Zhou, Guoli et al. (2011) Deficiencies in acetyl-CoA carboxylase and fatty acid synthase 1 differentially affect eggshell formation and blood meal digestion in Aedes aegypti. Insect Biochem Mol Biol 41:946-55
Zhou, Guoli; Miesfeld, Roger L (2009) Energy metabolism during diapause in Culex pipiens mosquitoes. J Insect Physiol 55:40-6
Stephens, Samuel B; Dodd, Rebecca D; Brewer, Joseph W et al. (2005) Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation. Mol Biol Cell 16:5819-31
Intine, Robert V; Tenenbaum, Scott A; Sakulich, Amy L et al. (2003) Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs. Mol Cell 12:1301-7
Lerner, Rachel S; Seiser, Robert M; Zheng, Tianli et al. (2003) Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes. RNA 9:1123-37
Figueroa, Angelica; Cuadrado, Ana; Fan, Jinshui et al. (2003) Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes. Mol Cell Biol 23:4991-5004
Atasoy, Ulus; Curry, Stephanie L; Lopez de Silanes, Isabel et al. (2003) Regulation of eotaxin gene expression by TNF-alpha and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR. J Immunol 171:4369-78
Eystathioy, Theophany; Chan, Edward K L; Tenenbaum, Scott A et al. (2002) A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol Biol Cell 13:1338-51

Showing the most recent 10 out of 11 publications