Fig 2: 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.

Fig 3: WTAP silencing alters BTV replication. (A–D) BUcEC were transfected with non-specific or a WTAP-specific siRNA. One day later, cells were infected with BTV8 (MOI = 0.01). After 24 h, relative WTAP mRNA and BTV-segment 1 levels were normalized to that of GAPDH (A and D, respectively). Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins (B) and the supernatants were titrated by determining the 50% tissue culture infective doses (TCID50)/mL (C). Experiments were achieved in triplicate (A,B,D) and with six individual samples for the viral titers (C). *, p-value (p) < 0.05; **, p < 0.005 and ***, p < 0.0005.

Fig 4: Comparative analysis of NS4 proteins from different BTV serotypes. (A) NS4 protein sequences from BTV8, BTV1, BTV4, BTV27, BTV25 and BTV30 were aligned with Clustal Omega version 1.2.3. (B) HEK-293T cells were transfected with expression vectors encoding GST alone or fused to BTV8-NS4, BTV1-NS4, BTV4-NS4, BTV27-NS4, BTV25-NS4 or BTV30-NS4 as indicated and tested for interaction with 3xFLAG fused to the full-length bovine WTAP. Total cell lysates were prepared at 48 h post-transfection (cell lysate; middle panel), and co-purifications of WTAP were assayed by pulldown using glutathione-Sepharose beads (pulldown; upper panel). Sizes are shown in kilodaltons (kDa).

Fig 5: Yeast two-hybrid mapping of BTV-NS4 and WTAP binding regions. (A) Fragments of BTV8-NS4 were generated by PCR using a matrix combination of specific primers and introduced into Gal4-BD vector by gap-repair in yeast cells expressing WTAP fused to Gal4-AD. (B) In a similar approach, fragments of WTAP were tested for the interaction with BTV8-NS4. (A,B) -L -W: Selective medium lacking leucine and tryptophan to control the gap-repair and yeast transformation. -L, -W, -H + 3-AT: selective medium lacking leucine, tryptophan and histidine and supplemented with 5 mM of 3-amino-triazole. Vertical and horizontal axes indicate the first and the last amino acid residues of each fragment tested, respectively. The WTAP and NS4 binding regions that have been identified are represented in lower diagrams.