Fig. 4: The m⁶A modification in the repeat-containing intron modulates DPR protein production.

a, Schematic of the bicistronic splicing reporter for (GGGGCC)₇₀ RAN translation, which mimics the splicing of C9 repeat RNA from the endogenous context. Predicted m⁶A sites were indicated as red lollipops in the intron sequence upstream of the repeats. b, Schematic of the monocistronic mRNA luciferase reporters for (GGGGCC)₇₀ RAN translation with or without the intronic sequence. Predicted m⁶A sites were indicated as red lollipops. c, Relative DPR levels were measured by luciferase assay upon METTL14 knockdown in the HeLa Flp-In cells expressing the reporters in (a). NLuc signal was normalized to the total protein level in each sample and the relative expression was compared to non-targeting siRNA control. Data are mean ± s.e.m. GA, ***p=0.0002; GR, ** p=0.0013; GP, ****p<0.0001, by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. d, Relative DPR levels were measured by luciferase assay upon METTL14 knockdown in the HeLa Flp-In cells expressing the reporters in (b). NLuc signal was normalized to the total protein level in each sample and the relative expression was compared to non-targeting siRNA control. Data are mean ± s.e.m. ****p<0.0001, by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. e, Western blotting of METTL14 in non-targeting or METTL14 siRNA transfected HeLa Flp-In cells. β-actin was blotted as internal control. f, RNA immunoprecipitation by IgG or m⁶A antibody (MeRIP) followed by RT-qPCR of HPRT1 RNA (left) or C9 repeat RNA (right) in the HeLa Flp-In C9R-NLuc reporter cells. HPRT1 RNA does not have m⁶A modification and serves as a negative control. Data are mean ± s.e.m. Points represent three biological replicates. C9R-NLuc, **p=0.0028; HPRT1, p=0.2584, by two-tailed t test. g, Timeline for the luciferase reporter experiments in human i³Neurons. h, Relative DPR levels were measured by luciferase assay upon METTL14 knockdown in the i³Neurons expressing the reporters as described in (g). n=3 biological replicates. Data are mean ± s.e.m. **p=0.001 and 0.0053, respectively, ****p<0.0001, by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Points represent three biological replicates in each group.