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- Title
Targeting mitochondrial and cytosolic substrates of TRIT1 isopentenyltransferase: Specificity determinants and tRNA-i<sup>6</sup>A37 profiles.
- Authors
Khalique, Abdul; Mattijssen, Sandy; Haddad, Alexander F.; Chaudhry, Shereen; Maraia, Richard J.
- Abstract
The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria. 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 base pair 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 is 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 anticodon identities of their tRNA substrates found throughout nature.
- Subjects
TRANSFER RNA; BASE pairs; EUKARYOTIC cells; GENETIC code; SCHIZOSACCHAROMYCES pombe; SACCHAROMYCES cerevisiae
- Publication
PLoS Genetics, 2020, Vol 16, Issue 4, p1
- ISSN
1553-7390
- Publication type
Article
- DOI
10.1371/journal.pgen.1008330