Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.
A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.
Warner
G J
GJ
Tufts University School of Medicine, Department of Physiology, Boston, Massachusetts 02111, USA.
Berry
M J
MJ
Moustafa
M E
ME
Carlson
B A
BA
Hatfield
D L
DL
Faust
J R
JR
D000818:Animals; D016466:CHO Cells; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D007453:Iodide Peroxidase / Q000235:genetics*; D007541:Isopentenyladenosine / Q000378:metabolism*; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D051140:Selenoproteins; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014981:Xenopus
D003062:Codon; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D051140:Selenoproteins; C112705:selenocysteinyl-tRNA; D007541:Isopentenyladenosine; D008148:Lovastatin; D007453:Iodide Peroxidase