Structure and function of the selenium translation element in the 3'-untranslated region of human cellular glutathione peroxidase mRNA.
In eukaryotes, incorporation of selenocysteine into the polypeptide chain at a UGA codon requires a unique sequence motif, or "selenium translation element" (STE), located in the 3'-untranslated region of the mRNA. The present study examines structure-function relationships of conserved sequence elements and of the putative stem-loop secondary structure in the STE of human GPX1 mRNA, which encodes the important antioxidant enzyme cellular glutathione peroxidase (EC 1.11.1.9). Deletion of the basal stem, upper stem, or apical loop of the stem-loop structure eliminated the ability of the STE to direct selenocysteine incorporation at the UGA codon of an epitope-tagged GPX1 reporter construct transfected into COS1 cells. However, mutations that change the primary nucleotide sequence of nonconserved portions of the stem-loop, but preserve its overall secondary structure, by inversion of apical loop sequences or exchange of 5' and 3' sides of stem segments, had little or no effect on selenocysteine incorporation. Effects of single- and double-nucleotide substitutions in three short, highly conserved elements in the GPX1 STE depended in large part on their computer-predicted perturbation of the stem-loop and its midstem bulge. Only in the conserved "AAA" apical loop sequence did mutations show major effects on function without predicted changes in secondary structure. Our results demonstrate the critical role of the three short, highly conserved sequences. However, outside of these elements, the function of the human GPX1 STE appears to depend strongly on the stem-loop secondary structure.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D017279:Selenocysteine / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D017279:Selenocysteine; D005979:Glutathione Peroxidase