Glutathione peroxidase represents the major cellular defense against H202 and derivative oxygen species, such as those produced by phagocytic leukocytes. The codon UGA usually serves as a """"""""stop"""""""" signal, but in glutathione peroxidase and other selenium-containing proteins it instead encodes the unusual amino acid selenocysteine. Conserved sequences in the 3'-untranslated region (3'UTR) of glutathione peroxidase mRNA are both necessary and sufficient for this alternative reading of the genetic code. This renewal application proposes to investigate the regulatory mechanisms and consequences of translational discrimination of the UGA codon in glutathione peroxidase mRNA from the standard UGA """"""""stop"""""""" codon in other transcripts. 1) The project will examine the role of the 3'UTR in the regulation of selenocysteine incorporation at the UGA codon in glutathione peroxidase gene transcripts. Deletion and substitution mutations will be utilized to determine structure-function relationships in a highly conserved sequence with a predicted stem-loop secondary structure. Synthetic stem-loops will test the function of identical overall secondary structures based on completely different primary nucleotide sequences. Fusion cDNAs constructed with coding sequences of non-selenoprotein genes (mutated to contain a UGA codon) will investigate whether the glutathione peroxidase 3'UTR will confer selenium regulation upon the mutated gene when expressed in stable transfectants and in transgenic mice. 2) RNA-binding proteins that recognize the 3'UTR stem-loop structure will be identified by gel-shift and UV crosslinking analysis. This information will provide the basis for molecular cloning of the RNA-binding proteins by direct screening of an expression library and by affinity purification of the proteins for microsequencing, followed by screening of cDNA libraries with derived oligonucleotide probes. 3) Experiments will determine whether the diminution in glutathione peroxidase gene transcripts in selenium deficiency reflects alterations in nuclear mRNA metabolism or in cytoplasmic mRNA stability. Examination of the mechanisms of regulation of glutathione peroxidase transcript levels by selenium will test the role of mRNA translation through the UGA codon as well as the effects of cis-acting sequences in the coding region and 3'UTR on mRNA levels and stability. The proposed studies should help elucidate the underlying regulatory mechanisms for the insertion of selenocysteine into glutathione peroxidase in leukocytes and, in addition, extend this knowledge to other cells and to the general issue of selenium regulation of protein synthesis and mRNA metabolism.
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