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Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue ofEscherichia coli MiaA,Saccharomyces cerevisiae Mod5,Schizosaccharomyces pombe Tit1, andCaenorhabditis 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 tRNASer AGA, tRNASer CGA, tRNASer 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 andS. 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 tRNASer UGA and tRNATrp UCA contained detectable i6A37. Moreover, while tRNASer levels were unaffected by TRIT1 knockdown, the tRNA[Ser]Sec UCA level was increased and the mt tRNASer UGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.

Human Cells Have a Limited Set of tRNA Anticodon Loop Substrates of the tRNA Isopentenyltransferase TRIT1 Tumor Suppressor