Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNA

The human AlkB family proteins, such as FTO and ALKBH5, are known to mediate RNA m⁶A demethylation. However, although ALKBH7 localizes in mitochondria and affects metabolism, the detailed biological function and mechanism have remained unknown for years. We developed Demethylation-Assisted Multiple Methylation sequencing (DAMM-seq) to simultaneously detect N¹-methyladenosine (m¹A), N³-methylcytidine (m³C), N¹-methylguanosine (m¹G) and N²,N²-dimethylguanosine (m²₂G) methylations in both steady-state RNA and nascent RNA, and discovered that human ALKBH7 demethylates m²₂G and m¹A within mt-Ile and mt-Leu1 pre-tRNA regions, respectively, in mitochondrial polycistronic RNA. DAMM-seq quantitatively and sensitively monitors the methylation stoichiometry change at pre-tRNA junctions within nascent mt-RNA, revealing the target region where ALKBH7 regulates RNA processing and local structural switch of polycistronic mt-RNAs. A new RNA demethylase in human cells was characterized through the base-resolution quantification of multiple RNA methylations in nascent mt-RNA, resolving the long-standing question about the functional substrate of ALKBH7.