Skip to main content
. Author manuscript; available in PMC: 2022 Jul 12.
Published in final edited form as: Nat Cell Biol. 2021 Jul 12;23(7):684–691. doi: 10.1038/s41556-021-00709-7

Extended Data Fig. 2 ∣. DAMM-seq reveals methylated sites in mitochondrial tRNA, and ALKBH7 does not demethylate m22G or other methylated bases (m1A, m3C, m1G) in mature mt-tRNAs.

Extended Data Fig. 2 ∣

a, Modification levels by LC-MS/MS in cellular small RNA fraction (input vs. engineered AlkB treatment) to showcase the demethylation efficiency and selectivity in DAMM-seq. P values = 0.0027, 0.0028, 0.4531, 0.0002, 0.6009, 0.5991, 0.5351, 0.5583 for methylated bases respectively; unpaired, two-tailed t-test. n = 3, biologically independent samples. Data are presented as mean values ± SD. For b-e, RT misincorporation frequency (input vs. engineered AlkB treatment) for: b, m1A9 in 14 mitochondrial tRNAs that contain m1A9; c, m3C32 in 2 mitochondrial tRNAs that contain m3C32; d, m1G9 in 5 mitochondrial tRNAs that contain m1G9; e, m1G37 in 4 mitochondrial tRNAs that contain m1G37. For b-e, n = 2 biologically independent samples. f, An example flowchart of library construction pipeline for DAMM-seq revealing methylation fraction change of specific m1A, m3C, m1G, and m22G sites in vivo, with altered TRMT1. g, TRMT1 knockdown efficiency in HeLa cells by mRNA level (below, normalized to GAPDH. n = 2 biologically independent samples) and protein level (above, compared to GAPDH). h, Mutation levels after RT for m22G26 in mt-tRNA in TRMT1-depleted HeLa cells vs. control. i, Mutation levels after RT for m22G26 in mt-tRNA in ALKBH7-depleted HepG2 cells vs. control. j, Mutation levels after RT for m22G26 in mt-tRNA in ALKBH7-overexpressed HeLa cells vs. control. For k-o, Mutation levels after RT (in ALKBH7-depleted HepG2 cells vs. control) for k, 3 mt-tRNAs with m1A58, l, 2 mt-tRNAs with m3C32, m, 14 mt-tRNAs with m1A9, n, 5 mt-tRNAs with m1G9, o, 4 mt-tRNAs with m1G37. For p-t, Mutation levels after RT (in ALKBH7-overexpressed HeLa cells vs. control) for p, 3 mt-tRNAs with m1A58, q, 2 mt-tRNAs with m3C32, r, 14 mt-tRNAs with m1A9, s, 5 mt-tRNAs with m1G9, t, 4 mt-tRNAs with m1G37. For h-t, n =2 biologically independent samples.