Abstract
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.
Highlights
Nucleoside modifications involving tRNAs at positions in or near the anticodon have been shown to affect any of a number of steps in the translation process [1]
One means of defining the physiological role of i6A on tRNA[Ser]Sec is to eliminate the adenosine at position 37, the site of isopentenylation
To determine if A37C-Sec tRNA was isopentenylated, we immunoprecipitated total RNA from cells transfected with either empty vector or A37C-XTSS with antiserum prepared in rabbits immunized with isopentenyladenosine [32] and performed Northern blots on the immunoprectipates
Summary
Vol 275, No 36, Issue of September 8, pp. 28110 –28119, 2000 Printed in U.S.A. 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. We conclude that isopentenylation of tRNA[Ser]Sec is required for efficient translational decoding of UGA and synthesis of selenoproteins. Both in vitro [12] and in vivo [13] evidence indicate that the i6A modification increases third position misreading due to decreased proofreading Together these results show that, in yeast and bacteria, isopentenylation at position 37 in tRNAs reading codons beginning with uridine has important physiologic effects on translation efficiency, especially with regard to tRNA suppression of stop codons. We examined the role of i6A at position 37 of tRNA[Ser]Sec on selenoprotein biosynthesis in vivo by generating Sec tRNAs lacking this modified base. The presence of i6A is important for efficient translation of Sec codons by tRNA[Ser]Sec
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