Mitochondria-specific RNA-modifying Enzymes Responsible for the Biosynthesis of the Wobble Base in Mitochondrial tRNAs

Abstract

Human mitochondrial (mt) tRNALys has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (m 5 s 2 U), at its anticodon wobble position. We previously found that the mt tRNA Lys , carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the m 5 s 2 U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.The precise codon-anticodon pairing that occurs during translation requires a post-transcriptional modification at the anticodon first position (the wobble position) of the tRNA (1-4). In mammalian mitochondrial (mt) 1 tRNAs, we recently discovered novel taurine-bearing modified uridines at the wobble position, namely, 5-taurinomethyluridine (m 5 U) in mt tRNA Leu(UUR) and 5-taurinomethyl-2-thiouridine (m 5 s 2 U) in mt tRNA Lys (5). We then showed that these wobble modifications are generated by the direct incorporation of taurine supplied by the medium. This is the first time it has been shown that taurine is a component of biological macromolecules.We have also shown that the taurine-bearing modifications do not occur in mutant mt tRNAs that contain pathogenic point mutations that are associated with mitochondrial encephalomyopathies (6, 7). Point mutations in mt tRNA genes are known to be responsible for a wide spectrum of human diseases that are caused by mitochondrial dysfunction. We found that the mt tRNA Leu(UUR) molecules obtained from pathogenic cells of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients (8), who have either the A3243G or the T3271C mutation, lack the m 5 U modification (6). To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery to construct a mt tRNA Leu(UUR) lacking the taurine modification but without the pathogenic mutation. This "operated" mt tRNA Leu(UUR) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNA Leu(UUR...

The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

“…In eukaryotes, the mitochondrial thiolase Mtu1 is clearly related to the bacterial thiolase MnmA (13,24), but, strikingly, it appears that its cytosolic counterpart, Ctu1, which we have identified in this study, is much more closely related to another bacterial thiolase, TtcA (6), which is involved in synthesis of 2-thiocytidine at position 32 in tRNAs, a modification absent in eukaryotes (see also the Introduction and ref. 14).…”

“…In line with a role of the thiolation in the tRNAs binding to their cognate codons, the absence of S 2 U in mitochondria results in impaired mitochondrial protein synthesis, which leads to reduced respiratory activity and human pathologies including MERFF (myoclonus epilepsy associated with ragged-red fibers), a subgroup of the mitochondrial encephalomyopathies where the mitochondrial tRNA LYS lacking the wobble modification cannot translate either of its codons (AAA and AAG) (13,(37)(38)(39).…”

“…S2), whereas the cytosolic thiolase was not identified (13). Recently, a new complex of two proteins from Thermus thermophilus (TtuA-TtuB for tRNAtwo-thiouridine) was identified as the thiolase of 2-thioribothymidine (S 2 U, also known as S 2 T) at position 54 of tRNA.…”

Taurine-containing Uridine Modifications in tRNA Anticodons Are Required to Decipher Non-universal Genetic Codes in Ascidian Mitochondria

“…4 Thus, m 5 (s 2 )U appears to be conserved in vertebrate mt tRNAs (23). m 5 (s 2 )U was not found in yeast and Caenorhabditis elegans mt tRNAs (25)(26)(27), which instead used cmnm 5 ( s 2 )U as the wobble modification. Due to the chemical similarity between 5-carboxymethylaminomethyluridine and m 5 U, both nucleotides are thought to be synthesized by a similar biosynthetic pathway, in which glycine is replaced with taurine in the latter case (23,27).…”

Human Mitochondrial Diseases Associated with tRNA Wobble Modification Deficiency

“…We have recently reported the identification and characterization of the tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. 17 To confirm that the A8344G mutation in mt tRNA Lys hinders the recognition of mt tRNA Lys by MTU1 and its subsequent formation of the 2-thio group of τm 5 s 2 U, it will be necessary to reconstitute in vitro the formation of 2-thio uridine in mt tRNA Lys using recombinant MTU1 with other factors. Nevertheless, the fact that all three mutant tRNAs possess a wobble modification deficiency prompted us to speculate that this RNA modification disorder may be a major molecular cause of the pathogenesis of both MELAS and MERRF.…”