© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. NATURE STRUCTURAL & MOLECULAR BIOLOGY ADVANCE ONLINE PUBLICATION  ARTICLES The ‘universal’ genetic code discovered in the 1960s was thought to be common to all living organisms and viruses. The apparent conserva- tion of the genetic code in many organisms led to the frozen-accident theory 1 , which assumes that all domains of life evolved from a single, closely interbreeding organism and that the genetic code established in that ancestor has remained fixed and immutable over the entire course of evolution. However, in 1979, the first non-universal genetic code was identified in the mitochondrial decoding system 2 . In human mitochondria, four codons deviate from the universal code: AUA, UGA and AGA/AGG (AGR), encoding methionine, tryptophan and STOP, respectively. The discovery of a non-universal genetic code that violated the frozen-accident theory lead to the alternative hypothesis that the genetic code is not fixed and instead has continued to evolve. To date, multiple instances of genetic code variation have been identi- fied in both nuclear and mitochondrial decoding systems from vari- ous organisms 3,4 , indicating that the diversity of the genetic code is larger than previously thought. Post-transcriptional modifications in the anticodon region of tRNAs play key roles in the alteration of the genetic code 4,5 . 5-Formylcytidine (f 5 C) at the first position of the anticodon (position 34) of mt-tRNA Met is required to decipher the AUA codon as methio- nine in the mitochondria of mammals 6,7 , nematodes 8 and insects (Drosophila melanogaster) 9 . We demonstrated that a lack of f 5 C34 results in severe reduction in mitochondrial protein synthesis and res- piratory activity 6,10 . In addition, 5-taurinomethyluridine (τm 5 U) and its 2-thiouridine derivative (τm 5 s 2 U) at the first anticodon position of mt-tRNAs contribute to the deciphering of multiple non-universal codons in mitochondria (AUA for methionine, UGA for tryptophan, and in tunicates, AGR for glycine) 4,11 . In echinoderm and some platyhelminth mitochondria, the AAA codon is interpreted as asparagine instead of lysine 12 (Fig. 1a). Thus, mt-tRNA Asn reads AAA in addition to AAU and AAC, and mt-tRNA Lys reads only AAG. Starfish (Asterias amurensis) mt-tRNA Asn has pseu- douridine (Ψ) at the middle of the anticodon (position 35) 13 . In vitro translation revealed that Ψ35 enables this tRNA to decode the AAA codon more efficiently than the unmodified form, thus suggesting that Ψ35 expands the decoding capability of mt-tRNA Asn . In the universal decoding system, in mt-tRNA Asn , the first position of the anticodon (position 34) is frequently queuosine (Q), which is required to strictly decipher AAY codons by preventing misreading of AAR codons 14 . Curiously, starfish mt-tRNA Asn lost Q34 because of the presence of C33, which acts as a strong negative determinant for the Q-inserting enzyme tRNA-guanine transglycosylase, indicating that the lack of Q34 in mt-tRNA Asn aids in the efficient decoding of the AAA codon 13 . Because accurate decoding is maintained by competition among near-cognate tRNAs, we need to consider the role of mt-tRNA Lys in establishing the alterative genetic code in echinoderm mitochondria. If mt-tRNA Lys had the ability to decode AAA, ambiguous assignment would destroy mitochondrial proteome integrity by causing an unac- ceptable amino acid substitution. Although mt-tRNA Lys possesses a CUU anticodon, which corresponds to an AAG codon, an unmodi- fied C34 is in general capable of pairing with A3 of the codon in the ribosomal A site. Escherichia coli tRNA Met with unmodified C34 rec- ognizes the AUA codon 15 ; accordingly, bacterial tRNA Met possesses 1 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan. 2 Department of Applied Life Sciences, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. 3 Deceased. Correspondence should be addressed to T.S. (ts@chembio.t.u-tokyo.ac.jp). Received 27 April; accepted 11 July; published online 7 August 2017; doi:10.1038/nsmb.3449 Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria Asuteka Nagao 1 , Mitsuhiro Ohara 1 , Kenjyo Miyauchi 1 , Shin-ichi Yokobori 2 , Akihiko Yamagishi 2 , Kimitsuna Watanabe 2,3 & Tsutomu Suzuki 1 The genetic code is not frozen but still evolving, which can result in the acquisition of ‘dialectal’ codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non- universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNA Lys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N 6 -threonylcarbamoyladenosine (ht 6 A), 3′ adjacent to the anticodon (position 37). Biochemical analysis revealed that ht 6 A37 has the ability to prevent mt-tRNA Lys from misreading AAA as lysine, thereby indicating that hydroxylation of N 6 -threonylcarbamoyladenosine (t 6 A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.