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.