TNFalpha is a pro-inflammatory cytokine produced transiently by several cell types in response to infection or injury. Production of TNFalpha is tightly regulated at the levels of transcription, mRNA decay and translation and aberrant expression of this potent molecule has been linked to autoimmune disorders, rheumatoid arthritis, Crohn's disease and other inflammatory conditions. The translational regulation of TNFalpha is mediated by AU-rich elements (AREs) located in the 3'-UTR of the mRNA. The ARE is involved in translational repression in resting cells as well as in activation of translation under stimulatory conditions. Several ARE-binding proteins have been identified and, of these, TIA-1 and TIAR have been implicated in translational repression. However, the factors involved in up-regulation of TNFalpha translation are not known. A system for studying this phenomenon has been developed in the yeast Saccharomyces cerevisiae. The yeast homologues of the ARE-binding protein Tristetraprolin (TTP) and a yeast homologue of TIA-1/TIAR were found to be key mediators of translation regulation by the ARE. The goals of this proposal are to utilize the yeast system to characterize the mechanism of translational regulation by the TNFalpha ARE, and to perform a genetic screen to identify the trans-acting factors required for accurate regulation. In addition, the role of TTP and its murine homologues (Tis 11 b and Tis 11 d) in modulating TNFalpha translation in mouse macrophages will be investigated. The experiments described here aim to increase our understanding of both general ARE function and regulation of TNFalpha expression.
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