Polysome distribution of phospholipid hydroperoxide glutathione peroxidasemRNA: Evidence for a block in elongation at the UGA/selenocysteine codon

The translation of mammalian selenoprotein mRNAs requires the 3' untranslat ed region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec), Sele noprotein biosynthesis is inefficient, which may be due to competition betw een Sec insertion and termination at the UGA/Sec codon, We analyzed the pol ysome distribution of phospholipid hydroperoxide glutathione peroxidase (PH GPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts, In linear sucrose gradients , the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn sup eroxide dismutase (SOD) mRNA, which has a coding region of similar size, Se lenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA, To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome di stribution of wild-type and mutant mRNAs translated in vitro, Mutation of t he UGA/Sec codon to UGU/cysteine increased ribosome loading and protein syn thesis. When UGA/Sec was replaced with UAA or when the SECIS element core w as deleted, the distribution of the mutant mRNAs was similar to the wild-ty pe mRNA, Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx m RNA, but had no effect on the mutant mRNAs, These results suggest that elon gation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this blo ck by promoting Sec incorporation instead of termination.