Fig. 6: The RNA-binding protein PSPC1 preferentially interacts with K235-acetylated ALKBH5.

a K235 is distant from the active site of ALKBH5. The relative position of K235 (magenta) with respect to the active site, is depicted by a collection of functionally important residues shown as green sticks. The covalent inhibitor IOX3 is shown in orange, and the active site manganese is shown as a sphere. The structure was drawn using the structure PBD 4NJ4. b PSPC1 was identified to preferentially interact with ALKBH5, and not its K235R mutant, based on the interactomics assay. The wild-type ALKBH5 or its mutant K235R plasmids together with the KAT8 vector were cotransfected into ALKBH5 KO HeLa cells, and the co-IPed wild-type ALKBH5 and its mutant K235R complexes were identified by mass spectrometry. c, d ALKBH5-FLAG (c) and PSPC1 (d) complexes were IPed in ALKBH5 KO HeLa cells stably re-expressing wild-type ALKBH5-FLAG or its mutants K235R or K235Q using anti-FLAG and PSPC1 antibodies, respectively, and the indicated proteins were detected. e The direct binding of recombinant GST-ALKBH5 to recombinant PSPC1-His was analyzed using a GST pull-down assay. f The PSPC1-HA plasmid together with the KAT8 plasmid were cotransfected into ALKBH5 KO HeLa cells stably re-expressing wild-type ALKBH5-FLAG or the K235R mutant. The PSPC1-HA complexes were IPed using anti-HA, and K235 acetylation and ALKBH5-FLAG were detected. g Recombinant GST-ALKBH5 was K235-acetylated by immunopurified KAT8, and the direct binding of recombinant K235-acetylated GST-ALKBH5 to recombinant PSPC1-His was analyzed using a GST pull-down assay. h The indicated ALKBH5-FLAG mutants were transfected into ALKBH5 KO HeLa cells, ALKBH5-FLAG complexes were IPed using an anti-FLAG antibody, and PSPC1 was detected. A total of 70–75ΔA and 70–75 Del indicate that aa 70–75 in ALKBH5 were mutated into AAAAAA and deleted, respectively. Source data are provided as a Source Data file.