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-) tRNA^Lys 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-tRNA^Lys 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.