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