The long-term objective of this project is to identify the mechanisms that regulate messenger RNA stability. Although much has been learned about transcriptional gene regulation, very little is known about the factors that control messenger RNA stability. It is increasingly recognized that many cytokine genes are regulated by changes in mRNA stability, but the mechanisms by which these mRNAs are stabilized or degraded in response to external stimuli have not been identified. In this project, the mechanisms of posttranscriptional regulation of a human inflammatory cytokine gene, gro, by interleukin-1 (IL-1) will be investigated. The minimum sequences that are necessary and sufficient to confer IL-1-dependent regulation of mRNA stability will be determined by transfection of altered gro genes. The functional significance of predicted secondary structural elements in the regulatory sequences will be tested by site-directed mutagenesis. Cytoplasmic factors that interact with the regulatory sequences will be characterized using an in vitro approach. The process of gro mRNA decay in vivo will be examined to determine the initial sites of nuclease attack and to identify pharmacologic agents that control gro mRNA turnover. An in vitro system will be developed to enable isolation of factors that regulate gro mRNA stability. This project will increase understanding of molecular events that control the inflammatory response and should elucidate a mechanism of gene regulation fundamental to biologic responses.
