Molecular basis for t(6)A modification in human mitochondria

N (6)-Threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNA(Thr) (hmtRNA(Thr)). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t(6)A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t(6)A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNA(Thr) to t(6)A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNA(Ile) or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNA(Ser)(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t(6)A modification levels.