Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.
The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) 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 superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium 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 distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.
Fletcher
J E
JE
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Copeland
P R
PR
Driscoll
D M
DM
D020413:3' Untranslated Regions / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002458:Cell Fractionation; D003062:Codon / Q000235:genetics; D004251:DNA Transposable Elements; D005979:Glutathione Peroxidase / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000235:genetics; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011132:Polyribosomes / Q000235:genetics*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology; D017279:Selenocysteine; D051140:Selenoproteins; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D003062:Codon; D004251:DNA Transposable Elements; D011506:Proteins; D012333:RNA, Messenger; D051140:Selenoproteins; D017279:Selenocysteine; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase