Fig. 4: PRMT1-mediated methylation at R251 is required for the oncogenic function of NONO.

a Flag-NONO protein were immunoprecipitated from control or PRMT1-silenced KM12 cells, separated by SDS–PAGE, and subjected to silver staining. b LC–MS/MS analysis of the NONO R251 methylation site. The fragmentation pattern of the typical NONO peptide EREQPPR is shown. Fragment ions are shown as b and y ions; ++ represents the loss of doubly charged ions. c R251K mutation abolishes PRMT1-mediated aDMA modification of NONO. NONO R-to-K mutants are illustrated schematically at the top. WT or R-to-K mutant NONO protein was immunoprecipitated with Flag beads and then immunoblotted with anti-ADMA antibody. d NONO is methylated by PRMT1 at R251, as determined with the in vitro methylation assay. GST-tagged GAR, NONO, and R251K were incubated with purified GST-PRMT1 in the presence or absence of 0.6 μM SAM. Proteins were separated on SDS–PAGE and subjected to western blotting analysis and Coomassie blue staining. GAR, glycine- and arginine-rich N-terminal region of fibrillarin. e NONO R251K mutation reduces cell proliferation. KM12 (1 × 10⁴) and HCT8 (2 × 10⁴) cells were seeded on day 0 and counted on days 2 and 4. NONO R251K mutation inhibited KM12 cell migration in the wound-healing assay (f) and invasion in the transwell assay (g). For experiments shown in e–g, KM12 cells were transfected with pCDH-CMV-Flag-NONO (WT) or pCDH-CMV-Flag-NONO-R251K (R251K mutant) for 24 h before the indicated assay. Scale bar, 400 μm. *P < 0.05, **P < 0.01, ***P < 0.001.