Human TRIT1 is a
tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to
adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in
codon-specific ways. TRIT1 is a
tumor suppressor whose mutant alleles are associated with
cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using
small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the
anticodon loop, only
tRNA(Ser)AGA,
tRNA(Ser)CGA,
tRNA(Ser)UGA, and
selenocysteine tRNA with UCA (
tRNA([Ser]Sec)UCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser,
tRNA([Ser]Sec)UCA is partially (∼40%) modified. Exogenous
selenium and other treatments that decreased the i6A37 content of
tRNA([Ser]Sec)UCA led to increased levels of the
tRNA([Ser]Sec)UCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs
tRNA(Ser)UGA and
tRNA(Trp)UCA contained detectable i6A37. Moreover, while
tRNA(Ser) levels were unaffected by TRIT1 knockdown, the
tRNA([Ser]Sec)UCA level was increased and the mt
tRNA(Ser)UGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.