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 (i⁶A). 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 i⁶A. 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.