ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria (mt) and affects metabolism, but its function and mechanism of action are unknown. Here, we report an approach to site-specifically detect m1A, m3C, m1G, and m22G modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates N2, N2-dimethylguanosine (m22G) and N1-methyladenosine (m1A) within mt-Ile and mt-Leu1 pre-tRNA regions, respectively, in nascent polycistronic mt-RNA. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mt-RNAs. Depletion of ALKBH7 leads to increased polycistronic mt-RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, as well as notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mt-RNA processing and mitochondrial activity. [dataset1] 4 samples (Sample 1-4): Duplicates for DAMM-seq of cellular small RNA samples from HepG2 cells, including RNA input and Engineered-AlkB-treated. [dataset2] 12 samples (Sample 13-24): Duplicates for DAMM-seq of mitochondrial tRNA samples to observe methylation fraction change in vivo, including siALKBH7 vs. siControl, shTRMT1 vs. shControl and o/e ALKBH7 vs. o/e Control in HepG2 and HeLa cell lines. [dataset3] 16 samples (Sample 25-40): Duplicates for DAMM-seq of mitochondrial dsRNA samples to observe methylation fraction change in vivo, including siALKBH7 vs. siControl, ALKBH7 wild-type vs. ALKBH7 knockout, shTRMT1 vs. shControl and o/e ALKBH7 vs. o/e Control in HepG2 and HeLa cell lines. [dataset4] 8 samples (Sample 41-48): Duplicates for icSHAPE of mitochondrial dsRNA samples to observe flexible regions within dsRNA in vivo, including input and pulldown samples for siALKBH7 vs. siControl in HepG2 cells. [dataset5] 6 samples (Sample 49-54): Duplicates for DAMM-seq of HepG2 dsRNA samples to observe methylation fraction change in vivo, including siALKBH7+empty vector, siALKBH7+ALKBH7-WT and siALKBH7+ALKBH7-mutant. [dataset6] 12 samples (Sample 55-66): Duplicates for DAMM-seq of HepG2 EU-labeled RNA samples to observe mt-mRNA and junction level change in vivo, including 10-min EU-labeled RNA from siALKBH7 vs. siControl, 10-min EU-labeled RNA from wild-type vs. ALKBH7 KO HepG2 cells, 20-min EU-labeled RNA from wild-type HepG2 cells and 30-min EU-labeled RNA from wild-type HepG2 cells.