Following T lymphocyte activation growth, regulatory genes including cytokine genes and proto-oncogenes are expressed for a defined period and then their expression is turned off, at least in part, through selective degradation of their mRNA transcripts. Many of these genes contain AU-rich elements (AREs) in their 3' untranslated region, which selectively target mRNA transcripts for degradation. The mechanism by which AREs mediate mRNA degradation has not been defined but appears to involve trans-acting proteins that bind to ARE sequences. The broad, long-term objective of this project is to understand the role of ARE-binding proteins in regulating the expression of T lymphocyte activation genes. The application focuses on characterizing the regulation and function of the ARE-binding proteins tristetraprolin (TTP) and HuA proteins in T lymphocytes. The first specific aim is to characterize the expression of ARE-binding proteins in T lymphocytes. Experiments will be performed to define the mechanism by which ARE-binding proteins are induced following T-cell activation. The second specific aim is to characterize the interaction of ARE-binding proteins with RNA. Experiments will be performed to evaluate the binding specificity and affinities of the TTP and HuA proteins for ARE sequences. The third specific aim is to evaluate the role of ARE-binding proteins in regulating mRNA degradation. The effect of these proteins on mRNA degradation will be examined using an in vitro RNA stability assay and by over-expression of these proteins in cell lines. Understanding the regulation and function of ARE-binding proteins will provide insights into cellular growth regulation and cancer. Since cytokines play critical roles in autoimmune diseases, cancer, and a variety of infectious diseases, a better understanding of the regulation of cytokine genes may provide new insights into the pathogenesis and treatment of these diseases.
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