Fig. 7: PSPC1 is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of RNA m⁶A by ALKBH5, thereby promoting RNA m⁶A removal by ALKBH5.

a PSPC1 KD increased cellular RNA m⁶A levels, whereas PSPC1 re-expression decreased cellular RNA m⁶A levels in PSPC1 KD cells. b HeLa cells were transfected with the indicated dose of PSPC1 plasmid, and cellular RNA m⁶A was determined. c Recombinant ALKBH5 was incubated with recombinant PSPC1 and m⁶A RNA oligos, and the m⁶A level was determined. d ALKBH5 plasmid together with anti-PSPC1 siRNA were cotransfected into ALKBH5 KO HeLa cells, and cellular RNA m⁶A was determined by dot blotting and LC‒MS/MS. e ALKBH5 plasmid together with PSPC1 plasmid were cotransfected into ALKBH5 KO HeLa cells, and cellular RNA m⁶A was determined by dot blotting and m⁶A ELISA. f Cumulative distribution curve for the abundance in NC, ALKBH5 KD and PSPC1 KD cells. g Distribution of m⁶A peaks in the 5′ UTR, CDS, stop codon and 3′ UTR between ALKBH5 KD-mediated m⁶A peaks and PSPC1 KD-mediated m⁶A peaks. h KAT8 plasmid together with anti-PSPC1 siRNA were cotransfected into ALKBH5 KO HeLa cells stably re-expressing the wild-type ALKBH5-FLAG or K235R mutant, and the cellular RNA m⁶A level was determined. i Immunopurified wild-type ALKBH5 or its K235R mutant was incubated with immunopurified PSPC1 and m⁶A RNA oligos, and the m⁶A level was determined. j The PSPC1 plasmid was transfected into ALKBH5 KO HeLa cells stably re-expressing wild-type ALKBH5-FLAG or K235R mutant, and the in vitro binding of wild-type ALKBH5 and K235R mutant to m⁶A-unmethylated or methylated RNA oligos was investigated. k The in vitro direct binding of recombinant ALKBH5 to m⁶A-unmethylated or methylated RNA oligos was investigated. l, m Anti-PSPC1 siRNAs with the wild-type sPSPC1 plasmid or the sPSPC1 mutant, which does not bind to RNAs, were cotransfected into ALKBH5 KO HeLa cells stably re-expressing wild-type ALKBH5-FLAG, and cellular RNA m⁶A was determined (l). The in vitro binding of ALKBH5 to m⁶A-methylated RNA oligos was investigated (m). Source data are provided as a Source Data file.