Fig. 1: m⁶A levels are enriched in the brain and are modulated with heat stress.

a Dot blot assay of polyA+ RNA m⁶A levels in fly heads, w¹¹¹⁸ (BL5905), was extracted immediately after HS at 38.5 °C for 10 min, 30 min, 1 h, and HS 1 and 24 h recovery. Quantification of m⁶A levels relative to basal (intensity = 1) and normalized to methylene blue. 1 ul dots of 200 and 100 ng polyA+ from the same replicate are blotted onto the membrane. n = 3 biological replicates, 200 heads per sample condition and replicate. Data presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001, two-way ANOVA with Sidak’s test. In a from left to right comparisons: p = 0.0459, p < 0.0001, p = 0.0066, p = 0.0314, p = 0.0034, p < 0.0001, p = 0.0314, p = 0.0138, p = 0.0296, ns not significant. b LC-MS/MS analysis of m⁶A/A% levels from head polyA+ RNA in basal and HS 30 min, in DaGal4>Mettl3 RNAi vs DaGal4 > mCherry RNAi conditions, n = 3 biological replicates for Mettl3 RNAi conditions, n = 4 for control animal conditions, 30 heads per replicate. Data presented as mean ± SD, *p < 0.05, ****p < 0.0001, one-way ANOVA with Tukey’s test. In b from left to right comparisons: p = 0.0266, p < 0.0001, ns not significant, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001. c LC-MS/MS analysis of m⁶A /A levels in polyA+ RNA from the brain in basal, 30 min HS, and Mettl3 RNAi in basal conditions. n = 3 biological replicates, 40 brains per replicate, DaGal4>Mettl3 RNAi vs mCherry RNAi. Data presented as mean ± SD, *p < 0.05, ***p < 0.01, ****p < 0.0001, one-way ANOVA with Tukey’s test. In c from left to right comparisons: p = 0.0377, p < 0.0001, p < 0.0001. d Dot blot of the whole fly w¹¹¹⁸ (BL5905) polyA+ RNA m⁶A levels with HS 30 min. Quantification of m⁶A levels normalized to control and methylene blue. n = 3 biological replicates, ten whole flies per sample condition and replicate. Data presented as mean, two-way ANOVA with Sikak’s test, ns not significant. e LC-MS/MS analysis of m⁶A /A levels in polyA+ RNA from brain, head, and whole fly of male flies w¹¹¹⁸ (BL5905). For brains n = 9 biological replicates with 40 brains per replicate, for heads n = 9 biological replicates with 30 heads per replicate, for whole flies n = 6 biological replicates with eight whole flies replicate. Data presented as mean ± SD, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s test. p = 0.0404, p < 0.0001, p = 0.0009. Additional details for fly genotypes for each figure are provided in Supplementary Data 7. Source data and statistical analysis are provided as a Source Data file.