ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

ALKBH1 is a 2-oxoglutarate-and Fe2+-dependent dioxygenase responsible for multiple cellular functions. Here, we show that ALKBH1 is involved in biogenesis of 5-hydroxymethyl-2'-O-methylcytidine (hm(5)Cm) and 5-formyl-2'-O-methylcytidine (f(5)Cm) at the first position (position 34) of anticodon in cytoplasmic tRNA(Leu), as well as f(5)C at the same position in mitochondrial tRNA(Me)t. Because f(5)C34 of mitochondrial tRNA(Met) is essential for translation of AUA, a non-universal codon in mammalian mitochondria, ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f(5) C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. We reconstituted formation of f(5)C34 on mitochondrial tRNA(Me)t in vitro, and found that ALKBH1 first hydroxylated m(5)C34 to form hm(5)C34, and then oxidized hm(5)C34 to form f(5)C34. Moreover, we found that the frequency of 1-methyladenosine (m(1)A) in two mitochondrial tRNAs increased in ALKBH1-knockout cells, indicating that ALKBH1 also has demethylation activity toward m1A in mt-tRNAs. Based on these results, we conclude that nuclear and mitochondrial ALKBH1 play distinct roles in tRNA modification.