Fig. 5. RNA methylation promotes Scratch1 mRNA splicing and export during neural differentiation.

a Sequence logo representing the consensus motif for m⁶A: RRm⁶ACH (R = A or G, G > A; H = A, U or C, U > A > C). b, In silico analysis of methylation probability in the different putative m⁶A sites present in Scratch1 pre-mRNA. c Schematic representation of the m⁶A-RIP protocol. d m⁶A-RIP-qPCR for Gapdh, Actb and Scratch1 at 0 h, 1 h and 6 h after the induction of differentiation (n = 3 biologically independent samples). e m⁶A-RIP-qPCR for Actb and Scratch1 at 0 h, 1 h and 6 h after the induction of differentiation: enrichment in Scratch1 levels compared to Actb (n = 3 biologically independent samples). f Schematic representation of the protocol used to induce the differentiation of NSCs in culture. g In situ hybridisation for Scratch1 mRNA (green) in control NSCs and in NSCs treated with 3-deazaadenosine (DAA). Quantification of mRNA distribution (n = 3 biologically independent samples). h In situ hybridisation for Scratch1 intron (red) in control NSCs and in NSCs treated with 3-deazaadenosine (DAA). Quantification of intron retention (n = 3 biologically independent samples). i Ratio of spliced Scratch1 mRNA in control and DAA-treated NSCs at 0 h, 1 h and 6 h after the induction of differentiation (control: p-value(1 h) = 0.0004, p-value(6 h) = 0.0001, DAA: p-value(1 h) = 0.0013, p-value(6 h)=0.0003, n = 3 biologically independent samples, by two-tailed Student’s t-test). j In situ hybridisation for Scratch1 mRNA (green) in control or Scratch1 overexpressing NSC cultures (±DAA) at 0 h, 1 h and 6 h after the induction of differentiation. Quantification of mRNA distribution (n = 3 biologically independent samples). k In situ hybridisation for Scratch1 intron (red) in control or Scratch1 overexpressing NSC cultures (±DAA) at 0 h, 1 h and 6 h after the induction of differentiation. Quantification of intron retention (n = 3 biologically independent samples). l Ratio of spliced Scratch1 mRNA in control or Scratch1 overexpressing NSC cultures (±DAA) at 0 h, 1 h and 6 h after the induction of differentiation (p-value(0 h) = 0.0001, p-value(1 h,C) = 0.3968, p-value(1 h,DAA) = 0.0002, p-value(6 h,C) = 0.2620, p-value(6 h,DAA) = 0.0001, n = 3 biologically independent samples, by two-tailed Student’s t-test). Scale bars represent 5 µm. Data are presented as mean values ± SEM. ns not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.