Fig 1: METTL3/14, but not WTAP, regulate m6A RNA at damage sitesa, U2OS cells stably expressing Flag-tagged METTL3 were microirradiated by UVA laser, incubated at 37°C for 2 min, and costained for m6A and Flag. The percentage of m6A-positive cells with colocalizing Flag signal is indicated on the right. Lower panel shows a representative cell with METTL3 localized at a damage site (arrow). b, Western blot for METTL3 in WT, METTL3 KO (M3 KO), or METTL3 KO U2OS cells stably expressing Flag-tagged METTL3 (M3 KO/WT-R) or its catalytic mutant (M3 KO/Cat-R). Actin is shown as a loading control. c, Western blot for METTL3 in U2OS cells expressing control (Control KD) or 2 independent shRNAs targeting METTL3 (M3 KD1, M3 KD2). Actin is shown as a loading control. d, WT or METTL3 KO (M3 KO) U2OS cells were irradiated or not (0′) with 15 J UVC, incubated at 37°C for 2 or 8 min, then subjected to poly(A)+ RNA extraction. Isolated poly(A)+ RNA was analyzed by dot-blot with an antibody recognizing m6A. Methylene blue staining was used as a loading control. e, Cells shown in (c) were subjected or not (0′) to 15 J UVC irradiation, incubated at 37°C for 1, 2, 4 or 10 min, and then subjected to poly(A)+ RNA extraction and dot-blot analysis as described in (d). f, WT cells were microirradiated by UVA laser, incubated at 37°C for 2 min, and stained for γH2A.X and METTL14. Arrows indicate representative γH2A.X-positive cells with colocalizing METTL14. The percentage of γH2A.X-positive cells showing colocalization with METTL14 is indicated on the right. g, WT U2OS cells expressing control (Control KD) or an shRNA targeting WTAP (WTAP KD) were subjected to UVA laser microirradiation, incubated at 37°C for 2 min, then costained for γH2A.X and WTAP, using three independent WTAP antibodies as indicated. The percentage of γH2A.X-positive cells displaying colocalizing WTAP signal is indicated on the right. h, Western blot for METTL14 in U2OS cells expressing control shRNA (Control KD) or shRNA targeting METTL14 (M14 KD). Actin is shown as a loading control. i, Western blot of METTL3 and METTL14 in WT, 2 independent METTL3 KO (M3 KO), or 2 independent METTL3 KO/METTL14 KD (M3 KO + M14 KD) U2OS cell lines. Actin is shown as a loading control. M3 KO1 and M3 KO1 + M14 KD1 were used for all subsequent experiments. j, WT, METTL3 KD (M3 KD), METTL14 KD (M14 KD), WTAP KD, METTL3 KO (M3 KO), or METTL3 KO/METTL14 KD (M3 KO/M14 KD) U2OS cells were microirradiated by UVA laser, incubated at 37°C for 2 min, and costained for m6A and γH2A.X. The percentage of γH2A.X-positive cells displaying colocalizing m6A signal is indicated on the right. All images are representative of at least 50 cells in triplicate. Scale bar, 20 μm.