ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation^1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism³, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N¹-methyladenosine (m¹A), N³-methylcytidine (m³C), N¹-methylguanosine (m¹G) and N²,N²-dimethylguanosine (m₂²G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m₂²G and m¹A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA^4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.