AN RNA-BINDING PROTEIN RECOGNIZES A MAMMALIAN SELENOCYSTEINE INSERTION-SEQUENCE ELEMENT REQUIRED FOR COTRANSLATIONAL INCORPORATION OF SELENOCYSTEINE

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocyst eine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated r egion (UTR). In this study, we investigated the SECIS elements and cel lular proteins required for selenocysteine insertion in rat phospholip id hydroperoxide glutathione peroxidase (PhGPx), We developed a transl ational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter, Insertion of a UGA or UAA codon int o the coding region of luciferase abolished luciferase activity, Howev er, activity was restored to the UGA mutant, but not to the UAA mutant , upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione pe roxidase (GPx) also allowed translational readthrough, whereas the PhG Px and GPx antisense 3' UTRs did not, Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the term inal loop) abolished readthrough activity, UV cross-linking studies id entified a 120-kDa protein in rat testis that binds specifically to th e sense strands of the PhGPx and GPx 3' UTRs, Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that b inding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein bindin g also prevented readthrough of the UGA codon. Our results suggest tha t the 120-kDa protein is a significant component of the mechanism of s elenocysteine incorporation in mammalian cells.