Fig. 6

HNRNPA1 arginine 194 methylation is required for its oncogenic function and recognition by ZMYND11. a Schematic diagram showing the protein ___domain organization of HNRNPA1. Shown are amino acid sequence conservation of the glycine-arginine-rich (RGG) motif with highlighted arginine (R) residues at the indicated position. RRM: RNA recognition motif. b Whole-cell lysates from HEK293T cells expressing V5-tagged ZMYND11 and T7-tagged HNRNPA1 or its mutant lacking the RGG-box were subjected to co-immunoprecipitation (co-IP) with anti-V5 antibody followed by immunoblotting. c The lysates from HEK293T cells expressing V5-tagged ZMYND11 and T7-tagged HNRNPA1 or the indicated mutant constructs, were immunoprecipitated with anti-V5 antibody followed by immunoblotting, and the reciprocal co-IP shown in the bottom. Representative images and quantification of colony-formation (d), cell proliferation (e), migration (f), and invasion (g) of 22Rv1 cells with stable knockdown of HNRNPA1 and rescue by co-transfection with the indicated constructs. Scale bar, 400 μm. Statistical significance was assessed using two-tailed Student’s t test. ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001. h His-tag pull-down assay investigating direct interactions between the MYND ___domain of ZMYND11 and HNRNPA1. i Pull-down assays between the MYND ___domain of ZMYND11 and the biotin-labeled peptides carrying R194 monomethylation (MMA) or symmetric demethylation (SDMA). j Bio-layer interferometry (BLI) binding assay of ZMYND11-MYND and HNRNPA1-SDMA-R194. k The 22Rv1 nuclear extract (NE) were affinity-purified using the indicated biotin-labeled RNAs, and the eluted proteins were detected using anti-HNRNPA1 and ZMYND11 antibodies. l 22Rv1 cells were co-transfected with ZMYND11-V5 and HNRNPA1-T7 plasmid and RNA affinity purification was performed as in a using biotin-labeled RNA E19 (50–68). m 22Rv1 cells were transfected with ZMYND11-V5 plasmid at the indicated doses and RNA affinity purification was performed as in (k) using biotin-labeled RNA E19. n 22Rv1 cells were transfected with the indicated HNRNPA1-truncated constructs or HNRNPA1 R194K mutant and RNA affinity purification was performed as in (k) using biotin-labeled RNA E19 (50–68)