Fig 2: ALKBH3-WT, but not ALKBH3-Mut extends the lifespan of Drosophila expressing expanded Q78 CAG repeats, and reduces the ratios of m1A/rA, but not m6A/rA in CAG repeat RNA.a, The mRNA level of wild-type human ALKBH3 (ALKBH3-WT) or its catalytically inactive mutant (ALKBH3-Mut) in Q78 Drosophila was measured by RT-qPCR (n = 6 biologically independent samples, 15 heads per sample). Data are mean ± s.d. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. ns, P > 0.05; **P = 0.0016; ****P < 0.0001; b, Drosophila lifespan was evaluated with co-expression of wild-type human ALKBH3 or its catalytically inactive mutant in Q78 Drosophila (n = 150 animals per survival curve). c-d, The levels of m1A (c) and m6A (d) in Q78 flies with expression of wild-type human or its catalytically inactive mutant. Data are mean ± s.d., and represent three biologically independent samples (30 heads per sample). Ten-day old male flies on RU486 food were used for the experiments. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. ns, P > 0.05; *P = 0.012 between Q78 and Q78 + ALKBH3-WT, and *P = 0.027 between Q78 + ALKBH3-WT and Q78 + ALKBH3-Mut in c. Source Data

Fig 3: m1A induces cytoplasmic mis-localization and aggregation of endogenous TDP-43.a, Representative images of U2OS cells expressing CAG repeat RNA along with ectopic expression of ALKBH3 or shRNA-mediated knockdown of TRMT61A. Scale bars, 10 μm. b, Quantification of area of TDP-43 foci in a. (CAG)22: n = 31; (CAG)38: n = 33; (CAG)22 + ALKBH3: n = 32; (CAG)38 + ALKBH3: n = 30; shControl: n = 33; shTRMT61A-1: n = 31; shTRMT61A-3: n = 32. c, Percentage of TDP-43 co-localized with CAG repeat RNAs. Images are representative of five or six independent frames for each condition. n = 5 for (CAG)22, (CAG)38, (CAG)22 + ALKBH3 and (CAG)38 + ALKBH3; n = 6 for shControl, shTRMT61A-1 and shTRMT61A-3. 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 = 0.020, **P = 0.0059 and ***P = 0.0009 between (CAG)22 and (CAG)38; ***P = 0.0003 between (CAG)38 and (CAG)38 + ALKBH3.Source Data

Fig 4: m1A enhances the ability of endogenous TDP-43 protein to partition into stress granules.a, CAG repeat RNA-mediated localization of endogenous TDP-43 into stress granules in U2OS cells with ectopic expression of ALKBH3 or knockdown of TRMT61A. b, FISH and immunofluorescence microscopy were performed to assess the co-localization between G3BP1 and CAG repeat RNAs in U2OS cells with ectopic expression of ALKBH3 or knockdown of TRMT61A. c, Percentages of TDP-43 co-localized with G3BP1. Images are representative of 5 to 7 independent frames for each condition: n = 5 for (CAG)22; n = 7 for (CAG)38 and (CAG)22 + ALKBH3; n = 6 for (CAG)38 + ALKBH3, shControl, shTRMT61A-1 and shTRMT61A-3. d, Percentages of G3BP1 co-localized with CAG repeat RNAs. Images are representative of 6 or 7 independent frames for each condition: n = 7 for (CAG)22, (CAG)38, (CAG)22 + ALKBH3, (CAG)38 + ALKBH3 and shControl; n = 6 for shTRMT61A-1 and shTRMT61A-3. 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. c, *P = 0.033 between (CAG)22 and (CAG)38; *P = 0.043 between (CAG)22 and (CAG)22 + ALKBH3; **P = 0.0030. d, **P = 0.0072. Scale bars, 10 μm.Source Data

Fig 5: m1A in CAG repeat RNA increases with repeat length and contributes to neurodegeneration in C. elegans.a–c, The m1A/rA ratios in ectopically expressed CAG repeat RNAs isolated from HEK293T cells (a; n = 3 biologically independent experiments), ectopically expressed (CAG)38 RNA isolated from HEK293T cells with ectopic co-expression of the indicated RNA demethylases or stable knockdown of m1A methyltransferases (b; n = 3 biologically independent experiments), and CAG repeat RNA from mouse brain (n = 11 and 12 biologically independent samples for striatum (str) and cortex (ctx) tissues, respectively), Drosophila head (n = 5 biologically independent samples, 10-day old males, 30 heads per sample) and C. elegans (n = 3 biologically independent experiments) (c). d,e, Fluorescence images of representative Q0, Q19 and Q67 nematodes, in Q67 nematodes with ectopic expression of wild-type human ALKBH3 (ALKBH3-WT) or its catalytically inactive mutant (ALKBH3-Mut) (d), and in Q67 nematodes with or without knockdown of W02A11.1 gene (e). f, Ratios of gaps in the nerve cord in Q67 worms with or without expression of human ALKBH3-WT and ALKBH3-Mut (Q0: n = 20 worms; Q19: n = 20 worms; Q67: n = 17 worms; Q67 + ALKBH3-WT: n = 20 worms; Q67 + ALKBH3-Mut: n = 15 worms), and in Q67 nematodes with or without knockdown of W02A11.1 gene (Q67 + vector RNAi: n = 22 worms; Q67 + W02A11.1 RNAi: n = 23 worms). The ratios of gaps were calculated by dividing the total length of gaps over the entire length of the nerve cord. Data are mean ± s.d. P values for the data of mouse brain and Drosophila head samples in c, and between Q67 + vector RNAi and Q67 + W02A11.1 RNAi in f were determined using two-tailed Student’s t-test; all other P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant (P > 0.05). b, *P = 0.021. c, **P = 0.0058 and ***P = 0.0007. ****P < 0.0001. Scale bars, 100 μm.Source Data