Abstract
In mammalian mitochondria, 22 species of tRNAs encoded in mitochondrial DNA play crucial roles in the translation of 13 essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. Following transcription, mitochondrial tRNAs are modified by nuclear-encoded tRNA-modifying enzymes. These modifications are required for the proper functioning of mitochondrial tRNAs (mt tRNAs), and the absence of these modifications can cause pathological consequences. To date, however, the information available about these modifications has been incomplete. To address this issue, we isolated all 22 species of mt tRNAs from bovine liver and comprehensively determined the post-transcriptional modifications in each tRNA by mass spectrometry. Here, we describe the primary structures with post-transcriptional modifications of seven species of mt tRNAs which were previously uncharacterized, and provide revised information regarding base modifications in five other mt tRNAs. In the complete set of bovine mt tRNAs, we found 15 species of modified nucleosides at 118 positions (7.48% of total bases). This result provides insight into the molecular mechanisms underlying the decoding system in mammalian mitochondria and enables prediction of candidate tRNA-modifying enzymes responsible for each modification of mt tRNAs.
Highlights
Mitochondria, organelles present in most eukaryotic cells, provide the chemical energy required by living cells in the form of adenosine triphosphate (ATP), which is synthesized by the electron transport chain and oxidative phosphorylation [1]
11 species of bovine mt tRNAs have been described in published work, along with the details of their posttranscriptional modifications: Ser(UCN) [33], Ser(AGY) [34,35], Phe [36], Arg [35], Gly [35], Ile [35], Met [8,37], Val [35], Trp [35], Glu [7] and Gln [7]
Unmodified fragments were identified by comparing the observed m/z values with the calculated m/z values deduced from the mt tRNAAla gene encoded in bovine mtDNA (GenBank accession number V00654)
Summary
Mitochondria, organelles present in most eukaryotic cells, provide the chemical energy required by living cells in the form of adenosine triphosphate (ATP), which is synthesized by the electron transport chain and oxidative phosphorylation [1]. Mitochondria contain their own genomic DNA, called mitochondrial (mt)DNA, and unique transcription and translation machinery that converts their genetic information into proteins. TRNAs responsible for two-codon sets ending in purines (NNR; N = any four nucleotides) have modified uridines (xm5s2U-type) at their wobble positions [5].
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