Fig 2: m1A triggers cytoplasmic mis-localization and aggregation of ectopically expressed EGFP-TDP-43.a, Representative images of U2OS cells co-expressing EGFP-TDP-43 and CAG repeat RNAs with or without ALKBH3 overexpression. b, Quantification results for the areas of EGFP-TDP-43 foci. (CAG)22: n = 69; (CAG)38: n = 69; (CAG)22 + ALKBH3: n = 68; (CAG)38 + ALKBH3: n = 66. Data are mean ± s.d., and represent three biologically independent experiments. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. c, RNA FISH for assessing the co-localization of EGFP-TDP-43 with CAG repeat RNAs. d, Percentages of CAG repeat RNAs co-localized with EGFP-TDP-43. Data are mean ± s.d. and represent three biologically independent experiments. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. **P = 0.0012 between (CAG)22 and (CAG)38, **P = 0.0033 between (CAG)22 and (CAG)22 + ALKBH3, **P = 0.0074 between (CAG)22 + ALKBH3 and (CAG)38 + ALKBH3; ***P = 0.0006 between (CAG)38 and (CAG)38 + ALKBH3, and ***P = 0.0002 between (CAG)38 and (CAG)38 + TDP-43-5FL. Scale bar, 10 μm. Source Data

Fig 3: OGD/R induces different m1A methylation modification patterns in neurons.A Overall abundance of m1A levels in cortical neuron transcripts and after OGD/R induction. The box limits represent the upper quartile, the median, and the lower quartile. The extremes represent the maximum and minimum values (p < 2.2e-16, Kruskal–Wallis test). B Representative analysis of the modified region of the m1A transcript. C Transcripts are m1A modified during OGD/R induction. D The proportion of transcripts carrying 1, 2, 3, 4, 5, and 6 or more peaks in neurons and after OGD/R induction. E The distribution and F density of the m1A peaks on each chromosome. G GC content in the m1A sequence and non-m1A sequence during OGD/R induction. G + C% represents the percentage of GC dinucleotides in the sequence. H qRT-PCR showed changes in the expression of the methylation-related enzymes Trmt6, Trmt61a, Trmt10c, Alkbh3, and Ythdf3 in mouse cortical neurons after OGD/R induction. I, J Western blot showed changes in the expression of the methylation-related enzymes Trmt6, Trmt10c, Alkbh3, and Ythdf3 in mouse cortical neurons after OGD/R induction. Data analysis was performed using Student’s t test. The data are presented as the means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. n.s. no significant difference.

Fig 4: Signatures of genetic variation of m1A regulators in hepatocellular carcinoma (HCC) cells. (A) Comparative analysis of m1A-related genes expressed in tumor and normal liver tissues from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC); (B) Frequency of CNV variation in TCGA-LIHC regulators. Column height represents CNV alteration frequency. Deletion frequency is indicated by green dots, and the amplification frequency is indicated by red dots; (C) Location of alterations in copy number variation (CNV) of m1A regulators on 23 chromosomes using the TCGA-LIHC cohort; (D) Analysis of 10 m1A regulator expression profiles in the TCGA-LIHC cohort by principal component analysis (PCA). On the basis of m1A regulator expression profiles, we identified two subgroups without intersection, indicating excellent differentiation between tumor samples and normal samples. Tumors are labeled in blue and normal samples in yellow; (E–N) Overall survival of subgroups of low m1A regulator expression and high m1A regulator expression, including TRMT6 (E), TRMT61A (F), TRMT61B (G), TRMT10C (H), ALKBH1 (I), ALKBH3 (J), YTHDF1 (K), YTHDF2 (L), YTHDF3 (M), and YTHDC1 (N) *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group; ns, not significant

Fig 5: Characterization of expression levels of m1A regulators in BLCA and control cell lines. (A) qRT-PCR was done to check the relative mRNA expression levels of TRMT6, TRMT61A, ALKBH1, and ALKBH3. GAPDH was used as endogenous control, and mRNA expression was normalized to SV-HUC1. qRT-PCR data represented as mean and SD, based on three independent experiments. Welch’s t-test, two tailed, was used to determine statistically significant differences compared to SV-HUC. TRMT6: T24 (** p = 0.003) TRMT61A: T24 (** p = 0.004), HT1376 (** p = 0.01), 5637 (p = * 0.038), SW780 (* p = 0.042). ALKBH1: T24 (**** p = <0.0001), 5637 (* p = 0.002) HT1197 (* p = 0.02, ALKBH3: HT1197 (** p= 0.002) (B) Protein expression of TRMT6, TRMT61A, ALKBH1, and ALKBH3 by western blot in BLCA cell lines versus SV-HUC1 control cell line, with GAPDH as loading control. Relative protein expression was normalized to GAPDH.