Post-transcriptional regulation plays a major role in modulating antioxidant enzyme (AOE) expression in animal models of oxygen toxicity and tolerance to hyperoxia. The goal of this grant is to understand the mechanisms controlling AOE RNA stability and translation into protein. The experiments will test the hypothesis that catalase and manganese superoxide dismutase (MnSOD) RNA 3' and 5' untranslated regions (UTRs) and their corresponding binding proteins affect RNA stability and/or translation and that these interactions are, in part, responsible for altering the cell's ability to withstand oxidant stress. There are three specific aims. 1. To determine the role of trans-acting catalase and MnSOD 3'UTR RNA-binding proteins in post-transcriptional regulation. We will purify and identify the 3'UTR RNA-binding proteins. For catalase RNA-binding protein (CAT-BP), we will confirm whether CAT-BP is TOAD-64 as suggested by preliminary data. To purify MnSOD RNA-binding protein we will use classical protein purification procedures, together with RNA-affinity chromatography. Once identified, we wil generate cDNAs, antisense oligonucleotides, and antibodies specific for the BP to examine their function and regulation. To ascertain function, we will use both cell-free in vitro and transfected cell culture in vivo systems. 2. To examine the role of catalase and MnSOD 5' UTRs and binding proteins in the regulation of RNA stability and translation. These studies are a natural extension of studies on the role of the 3'UTR and will employ procedures analogous to those used in examining 3'UTR RNA-BPs. 3. To determine how MnSOD translation is regulated by the 3' and 5' UTRs in three animal paradigms: a) O toxicity caused by hyperoxic exposure of adult rats, b) O2 toxicity caused by pertussis toxin treatment of air-breathing rats, and c) protection against O2 toxicity afforded by lipopolysaccharide (LPS) treatment. By understanding the mechanism responsible for the block in MnSOD translation leading to O2 toxicit and the ability of pharmacological intervention (LPS) to overcome the block, w hope to understand how MnSOD protein expression can be increased to protect th lung under conditions of oxidant stress. In addition, because gene therapy studies are often directed at increasing gene dosage it is important to understand how to optimize RNA stability and translational efficiency to prevent a situation in which sufficient RNA is provided but not fully utilized in the production of protein.
