Extended Data Fig. 1: Human ALKBH7 effect on mt-RNA methylations by mass spec and the development of DAMM-seq to detect m1A, m3C, m1G and m22G in one sequencing run via misincorporation signature.
From: ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing
a, An overlay of crystal structure of E. coli AlkB and human ALKBH7 on three peripheral loops (two are identical as EcAlkB, one is the ALKBH7 specific loop). b, The unique active site of ALKBH7 resembles an engineered AlkB protein (D135S/L118V) that catalyzes demethylation of m22G. c, Alkbh7 mRNA levels (normalized to mouse Actb) in several mouse tissues. n = 3 biologically independent samples; data are presented as mean values ± SD. d, ALKBH7 mRNA levels (normalized to ACTB) in several human cancer cell lines. e, ALKBH7 knockdown efficiency in HepG2 cells by protein level (compared to β-tubulin). f, ALKBH7 knockdown efficiency in HepG2 cells by mRNA level (normalized to β-actin). For d and f, n = 2 biologically independent samples. g, Modification levels (m22G/G, m1G/G, m6A/A, m1A/A, m7G/G, m2G/G) by LC-MS/MS for mitochondrial tRNA in ALKBH7-depleted HepG2 cells vs. control. Unpaired, two-tailed t-test; n = 4 biologically independent samples; data are presented as mean values ± SD. h, Modification levels by LC-MS/MS for mitochondrial mRNA in ALKBH7-depleted HepG2 cells vs. control. i, Modification levels by LC-MS/MS for mitochondrial rRNA in ALKBH7-depleted HepG2 cells vs. control. For h-i, unpaired, two-tailed t-test; n = 3 biologically independent samples; data are presented as mean values ± SD. j, A flowchart of DAMM-seq library construction pipeline, detecting four types of methylated bases (m22G, m1G, m1A, m3C) by misincorporation signatures in a “one-pot” manner with or without engineered AlkB treatment. k, IGV plot for visualizing the mutation level drop down to zero after demethylase treatment in DAMM-seq.
