Selenoproteins are proteins carrying the rare amino acid Sec (selenocysteine). Full expression of selenoproteins requires modification of tRNA[Ser]Sec, including N6-isopentenylation of base A37. We show that Trit1 is a dimethylallyl:tRNA[Ser]Sec transferase. Knockdown of Trit1 reduces expression of selenoproteins. Incubation of in vitro transcribed tRNA[Ser]Sec with recombinant Trit1 transfers [14C]dimethylallyl pyrophosphate to tRNA[Ser]Sec. 37A>G tRNA[Ser]Sec is resistant to isopentenylation by Trit1.

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