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Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria


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-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3′ adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

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Figure 1: Discovery of new derivative of t6A in M. nudus mt-tRNALys.
Figure 2: Identification of ht6A.
Figure 3: Functional roles of t6A37 and ht6A37 in AAR decoding.


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We are grateful to the Suzuki laboratory members for many fruitful discussions and technical advice. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan (Japan Society for the Promotion of Science grant nos. 26113003, 26220205 and 24370093 to T.S., 26116003 to A.N., 24370093 to S.Y. and 24370093 to K.W.).

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Authors and Affiliations



A.N. and M.O. mainly performed the series of experiments. K.M. assisted with the biochemical work. S.Y., A.Y., and K.W. prepared RNA specimens. All authors discussed the results. T.S. and A.N. wrote the paper. T.S. designed and supervised all of the work.

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Correspondence to Tsutomu Suzuki.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Isolation of mt-tRNAs and RNA-MS analysis of mt-tRNAAsn from M. nudus

(a) M. nudus mt-tRNAs were isolated by RCC and resolved by 10% denaturing PAGE. The gel was stained with SYBR Gold (Invitrogen) and visualized using an FLA-7000 image analyzer (Fujifilm). The tRNAs as indicated by arrows were cut out of the gel and purified.

(b) Top panel: base peak chromatogram (BPC) of RNase T1-digested fragments; second and third panel: extracted ion chromatograms (XIC) for the negative ions of the anticodon-containing fragments as indicated. Molecular mass of each fragment numbered on the BPC is listed in Supplementary Table 1. Bottom panel shows the CID spectrum of the cyanoethylated anticodon-containing fragment. The c- and y-series product ions are indicated on the CID spectrum and assigned on the corresponding sequence.

(c) Secondary structure of M. nudus mt-tRNAAsn with post-transcriptional modifications. The numbering system of tRNA is based on the tRNA database (Juhling, F. et al., Nucleic Acids Res. 37, D159-62, 2009). m1A: 1-methyladenosine, m2,2G: N2, N2-dimethylguanosine, Ψ: pseudouridine, and t6A: N6-threonylcarbamoyladenosine.

Supplementary Figure 2 RNA-MS analysis of M. nudus mt-tRNALys

XICs of the anticodon-containing fragments with N428 at position 37 (upper panel), t6A37 (lower panel). Sequence, m/z values, and charge state of each fragment are indicated on the right side. The frequency of each modification was calculated from the ratio of the peak areas of the two fragments.

Supplementary Figure 3 CID spectrum of t6A base

The t6A base was generated by in-source fragmentation of t6A nucleoside in individual E. coli tRNAThr4. The product ions are assigned on the chemical structure of the t6A base. The internal fragment of the threonine moiety is indicated by a dotted line.

Supplementary Figure 4 Enzymatic synthesis of Thr and 4-hydroxythreonine

LtaE catalyzes an aldol condensation reaction of acetaldehyde and glycine to synthesize Thr utilizing pyridoxal phosphate (PLP) as a cofactor. LtaE catalyzes the same reaction with glycolaldehyde and glycine to form 4-hydroxythreonine.

Supplementary Figure 5 RPC-LC/MS co-injection analysis of the synthetic ht6A and natural N428

XICs of synthetic ht6A (left panel), nucleosides of M. nudus mt-tRNALys (middle panel), and co-injection of both samples (right panel). Lower panels show XICs of m1A (m/z 298) as controls.

Supplementary Figure 6 CID analysis of the synthetic ht6A

CID spectra of ht6A nucleoside (upper panel) and its base ion (lower panel). ht6A base (BH2+) was generated by in-source fragmentation of ht6A nucleoside of E. coli tRNALys transcript bearing ht6A37. The product ions are assigned in the corresponding chemical structures. The internal fragment of the methyl threonine moiety is indicated by a dotted line.

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Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 2 (PDF 831 kb)

Life Sciences Reporting Summary (PDF 1169 kb)

Supplementary Table 1

RNase T1-digested fragments of M. nudus mt-tRNAs detected by RNA-MS (XLSX 11 kb)

Exact molecular mass (Da), and observed and calculated monoisotopic m/z value with its charge state for each fragment are listed.

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Nagao, A., Ohara, M., Miyauchi, K. et al. Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria. Nat Struct Mol Biol 24, 778–782 (2017).

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