Targeting of mitochondrial and cytosolic substrates of tRNA isopentenyltransferases: selection of differential tRNA-i6A37 identity subsets

ABSTRACT tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of adenosine-37 (i6A37) of certain tRNAs, are among a minority of modification enzymes that act on both cytosolic and mitochondrial substrates. The Caenorhabditis elegans mitochondrial IPTase impacts life expectancy, and pathogenic mutations to human IPTase (TRIT1) that decrease i6A37 levels cause mitochondrial insufficiency and neurodevelopmental disease. Understanding of IPTase broad function should consider the differential identities of the tRNAs selected for i6A37 formation and their cognate codons, which vary among species in both their nuclear- and mitochondria-encoded tRNAs. Substrate selection is principally by recognition of the A36-A37-A38 sequence but can be negatively impacted by certain anticodons, and by ill-defined properties of the IPTase. Thus, tRNAs-i6A37 comprise a modification code system whose principles are incompletely understood. While Saccharomyces cerevisiae uses alternative translation initiation to target IPTase to mitochondria, our analyses indicate that TRIT1 uses a single initiation site to produce a mitochondrial targeting sequence (MTS) that we demonstrate by point mutagenesis using GFP imaging in human cells. We also examined cytosolic and mitochondrial tRNA modification by TRIT1 in Schizosaccharomyces pombe using tRNA-mediated suppression and i6A37-sensitive northern blotting. The TRIT1 MTS mutations indeed decrease mitochondrial-tRNA modification in S. pombe. We also show TRIT1 modification deficiency specific for tRNATrpCCA despite A36-A37-A38, consistent with the negative effect of the CCA anticodon as was described for Mod5 IPTase. This TRIT1 deficiency can be countered by over-expression. We propose a model of tRNA-i6A37 identity selection in eukaryotes that includes sensitivity to substrates with YYA anticodons. AUTHOR SUMMARY tRNA isopentenyltransferases (IPTases) are tRNA modification enzymes that are conserved in bacteria and eukaryotes. They add an isopentenyl group to the Adenosine base at position 37, adjacent to the anticodon of specific subsets of tRNAs that decode codons that begin with Uridine. This modification stabilizes the otherwise weak adjacent codon-anticodon basepair and increases the efficiency of decoding of the corresponding codons of the genetic code. IPTases belong to a group of enzymes that modify both cytoplasmic and mitochondrial tRNAs of eukaryotic cells. Interestingly, during evolution there were changes in the way that IPTases are targeted to mitochondria as well as changes in the relative numbers and identities of IPTase tRNA substrates in the cytoplasm vs. mitochondria, the latter consistent with phenotypic consequences of IPTase deficiencies in fission and budding yeasts, and mammals. Pathogenic mutations to human IPTase (TRIT1) cause mitochondrial insufficiency and neurodevelopmental disease, principally due to decreased modification of the mt-tRNA substrates. In this study, we identify the way human TRIT1 is targeted to mitochondria. We also show that TRIT1 exhibits a tRNA anticodon identity-specific substrate sensitivity. The work leads to new understanding of the IPTases and the variable codon identities of their tRNA substrates found throughout nature.