Published online 30 January 2018 Nucleic Acids Research, 2018, Vol. 46, No. 4 1565–1583 doi: 10.1093/nar/gky068 NAR Breakthrough Article Metabolic and chemical regulation of tRNA modification associated with taurine deficiency and human disease Kana Asano1,†, Takeo Suzuki1,*,†, Ayaka Saito1, Fan-Yan Wei2, Yoshiho Ikeuchi3, Tomoyuki Numata4, Ryou Tanaka5, Yoshihisa Yamane5, Takeshi Yamamoto6, Takanobu Goto7, Yoshihito Kishita8,KeiMurayama9, Akira Ohtake10, Yasushi Okazaki8,11, Kazuhito Tomizawa2, Yuriko Sakaguchi1 and Tsutomu Suzuki1,* 1Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 2Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan, 3Institute of Industrial Science, University of Tokyo, Meguro-ku, Tokyo 153-8505, Japan, 4Biological Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan, 5Department of Veterinary Surgery, Tokyo University of Agriculture and Technology, Animal Medical Center, Fuchu, Tokyo 183-8509, Japan, 6Tamaki Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Tamaki, Mie 519-0423, Japan, 7Department of Chemistry & Biochemistry, National Institute of Technology, Numazu College, Numazu, Shizuoka 410-8501, Japan, 8Division of Functional Genomics & Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama 350-1240, Japan, 9Department of Metabolism, Chiba Children’s Hospital, Midori-ku, Chiba 266-0007, Japan, 10Department of Pediatrics, Saitama Medical University, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan and 11Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama 350-1240, Japan Received December 05, 2017; Revised January 17, 2018; Editorial Decision January 18, 2018; Accepted January 23, 2018 ABSTRACT logical consequences. Taurine starvation resulted in downregulation of ␶ m5U frequency in cultured cells Modified uridine containing taurine, 5-taurinometh and animal tissues (cat liver and flatfish). Strikingly, yluridine (␶ m5U), is found at the anticodon first 5-carboxymethylaminomethyluridine (cmnm5U), in position of mitochondrial (mt-)transfer RNAs (tR- which the taurine moiety of ␶ m5U is replaced with NAs). Previously, we reported that ␶ m5U is absent glycine, was detected in mt-tRNAs from taurine- in mt-tRNAs with pathogenic mutations associated depleted cells. These results indicate that tRNA mod- with mitochondrial diseases. However, biogenesis ifications are dynamically regulated via sensing of and physiological role of ␶ m5U remained elusive. intracellular metabolites under physiological condi- Here, we elucidated ␶ m5U biogenesis by confirm- tion. ing that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for ␶ m5U formation cat- alyzed by MTO1 and GTPBP3. GTPBP3-knockout INTRODUCTION cells exhibited respiratory defects and reduced mi- tochondrial translation. Very little ␶ m5U34 was de- RNA molecules contain a wide variety of chemical mod- ifications that are introduced post-transcriptionally. RNA tected in patient’s cells with the GTPBP3 mutation, ␶ 5 modifications confer chemical diversity on simple RNA demonstrating that lack of m U results in patho- molecules, endowing them with a wider range of biological *To whom correspondence should be addressed. Tel: +81 3 5841 8752; Fax: +81 3 5841 0550; Email: [email protected] Correspondence may also be addressed to Takeo Suzuki. Tel: +81 3 5841 1260; Fax: +81 3 5841 0550; Email: t [email protected] †These authors contributed equally to the paper as first authors. C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 1566 Nucleic Acids Research, 2018, Vol. 46, No. 4 functions. To date, >130 species of RNA modifications have normally, but failed to efficiently decode the UUG codon been reported in various RNA molecules from all domains efficiently (22), strongly suggesting that inefficient decod- of life (1–4). Many of these modifications were discovered ing by the hypomodified tRNA results in defective trans- in transfer RNAs (tRNAs), and tRNA modifications are lation of protein subunits of respiratory chain complexes. involved in various molecular and physiological functions In addition, ␶m5s2U34 is absent from mt-tRNALys with (5,6). In particular, tRNA modifications at the first (wobble) the A8344G point mutation associated with MERRF (my- position of the anticodon (position 34) play critical roles in oclonus epilepsy associated with ragged red fibers) (23,24). regulating the codon–anticodon interaction at the A-site of The hypomodified mt-tRNALys also lacks the ability to de- ribosome during the decoding process in translation (7). code its cognate codons, leading to defective mitochondrial The mitochondrion is a eukaryotic intracellular organelle translation and respiratory activity (25). The pathogenic that produces adenosine triphosphate (ATP) by oxida- point mutations associated with MELAS and MERRF are tive phosphorylation (OXPHOS). Mitochondria contain thought to prevent recognition by the tRNA-modifying en- their own genome, called mitochondrial DNA (mtDNA), zymes responsible for ␶m5U formation. These findings rep- which encodes 13 essential subunits of the respiratory chain resent the first instance of human disease caused by aberrant complexes, two mt-rRNAs and 22 mt-tRNAs. The mam- RNA modifications (19). Hence, we propose ‘RNA mod- malian mitochondrial genetic code deviates from the uni- opathy’ as a distinct category of human disease. versal code by using four dialectal codons: AUA for Met, A metabolic labeling experiment revealed that extracel- UGA for Trp and AGA and AGG (AGR; R = AorG) lular taurine supplied in the medium is directly incorpo- for the stop signal. The 22 species of mt-tRNAs partici- rated to 5-taurinomethyl groups in mt-tRNAs (17). How- pate in translation of 13 proteins encoded in the mtDNA. ever, a comprehensive understanding of ␶m5U biogenesis RNA modifications in mt-tRNAs play critical roles inac- remains elusive. Based on a genetic study in yeast (26), we curate and efficient translation in mitochondria (8). Previ- previously predicted the tRNA modifying enzymes respon- ously, we mapped all modifications in 22 species of mam- sible for ␶m5s2U34. MTO1 and GTPBP3 are likely to be in- malian mt-tRNAs, and found 15 species of modified nucle- volved in the formation of the 5-taurinomethyl group, while osides at 118 positions (9). Three of the modifications, 5- MTU1 is responsible for the 2-thio group of ␶m5s2U34 in formylcytidine (f5C), 5-taurinomethyluridine (␶m5U) and mt-tRNAs. Genetic mutations in MTU1 were identified in 5-taurinomethyl-2-thiouridine (␶m5s2U) are specific to mt- patients of reversible infantile liver failure (RILF) (27–29). tRNAs. Liver-specific knockout of Mtu1 mice exhibited severe im- f5C is present at the first position of the anticodon in pairment of mitochondrial translation and respiratory ac- mt-tRNAMet (10), and f5C34 plays an essential role in tivities in the hepatocytes, demonstrating that mitochon- translation of the dialectal AUA codon as Met (8,11,12). drial dysfunction due to loss of 2-thiolation of ␶m5s2U34 is We and other groups, recently demonstrated that f5C34 a primary cause of RILF (30). Regarding 5-taurinomethyl is synthesized via consecutive reactions, initiated by 5- modification, exome sequencing identified pathogenic mu- methylcytidine (m5C) formation catalyzed by NSUN3 tations in both MTO1 (31–33)andGTPBP3 (34,35) associ- (13,14), followed by hydroxylation and oxidation of m5Cto ated with hypertrophic cardiomyopathy and lactic acidosis, generate f5C, mediated by ALKBH1 (15,16). Both enzymes indicating that lack of taurine modification results in mi- are required for efficient mitochondrial translation and res- tochondrial dysfunction via a mechanism similar to those piratory activity. We also identified two pathogenic point of MELAS and MERRF. However, it has not been directly mutations in mt-tRNAMet that impair f5C34 formation demonstrated that the frequency of ␶m5(s2)U34 is reduced (13). In addition, loss-of-function mutations in NSUN3 in patients’ cells or tissues. were detected in a patient with symptoms of mitochondrial In regard to the biogenesis of ␶m5U34, we demon- disease (14). Together, these observations indicate that loss strate here that the ␤-carbon of L-serine is a source of of f5C34 results in pathological consequences. the methylene group of ␶m5(s2)U34 via 5,10-methylene- Two taurine-containing uridines are present at the wob- tetrahydrofolate (THF) in one-carbon (1C) metabolism. ble positions of five mt-tRNAs: ␶m5U in the mt-tRNAs for This finding is supported by the observation that the Leu(UUR) and Trp, and ␶m5s2U in the mt-tRNAs for Glu, level of ␶m5(s2)U34 was reduced in cells harboring muta- Lys and Gln (9,17)(Figure1A). These modifications pro- tions in serine hydroxymethyltransferase 2 (SHMT2) and mote accurate decoding of purine-ending codons