Fig. 4: ALKBH5 acetylation at K235 is critical for the RNA m6A demethylation activity of ALKBH5.
a, b K235 acetylation of ALKBH5 decreased the cellular mRNA m6A levels. The wild-type ALKBH5 and its mutant K235R and K235Q plasmids were transfected into ALKBH5 KO HeLa cells, and the cellular mRNA m6A level was determined by dot blotting (a) and quantified by LC‒MS/MS analysis (b) (n = 3, two-tailed unpaired Student’s t test, mean ± SD). c, d Wild-type ALKBH5, but not the K235R mutant, directly demethylated m6A in the m6A-RNA oligos in the in vitro demethylation reaction. Immunopurified wild-type ALKBH5 and its mutant K235R (c) or recombinant wild-type ALKBH5 and its mutant K235R (d) were incubated with m6A RNA oligos; the m6A level was determined by dot blotting or LC‒MS/MS assays. e Cumulative distribution curve for the m6A peak abundance in NC, WT and K235R cells. f Distribution of m6A peaks in the 5′ UTR, CDS, stop codon and 3′ UTR in NC, WT and K235R cells. g Top consensus motif identified by HOMER with m6A peaks in NC, WT and K235R cells. h The indicated ALKBH5 vectors together with the KAT8 plasmid were cotransfected into ALKBH5 KO HeLa cells, and the cellular RNA m6A level was determined. i, j Recombinant wild-type ALKBH5 or its K235R mutant was incubated with m6A RNA oligos after recombinant wild-type ALKBH5 or its K235R mutant was treated with immunopurified KAT8 (i) or immunopurified HDAC7 (j), and the m6A level was determined. Source data are provided as a Source Data file.
