Fig. 1. X-chromosomal transcripts are more stable upon m⁶A depletion.

a, Experimental setup for the SLAM-seq experiment. b,c, Transcripts (n = 7,310) in control (b) and m⁶A-depleted conditions (c) show a median half-life (t_1/2) of 3.2 h and 3.5 h, respectively (P value = 5.25 × 10^–29, two-tailed Wilcoxon signed-rank test). Median s⁴U content for all transcripts is shown in black. d, Transcripts with m⁶A sites have significantly shorter half-lives (P value = 2.17 × 10^–18, two-tailed Wilcoxon rank-sum test). Cumulative fractions of transcripts with given half-lives for transcripts with (n = 2,342, green) or without (n = 4,967, black) m⁶A sites. e, Transcripts with m⁶A sites (n = 2,342) significantly increase in half-life upon m⁶A depletion (8% median increase, P value = 1.07 × 10^–61, two-tailed Wilcoxon signed-rank test), unmethylated transcripts (n = 4,967) were largely unaffected (0.3% median decrease, P value = 3.186 × 10^–5) (same gene set in both conditions). The mean half-life in each group is shown as a red dot. Boxes represent quartiles, center lines denote medians, and whiskers extend to most extreme values within 1.5 × interquartile range. f, Half-lives of autosomal transcripts significantly increase upon m⁶A depletion (P value = 3.03 × 10^–31, two-tailed Wilcoxon signed-rank test), while X-chromosomal transcripts remain unchanged (P value = 0.2121, two-tailed Wilcoxon signed-rank test). Distribution of half-lives for autosomal (n = 7,069) and X-chromosomal transcripts (n = 241) (same gene set in both conditions). The mean half-life in each group is shown as a red dot. Boxes are as in e. g, Median fold change (FC) in mRNA half-lives (log₂) for each chromosome in m⁶A-depleted (STM2457) over control (DMSO) conditions. X-chromosomal transcripts show the lowest half-life increase upon m⁶A depletion (P value = 0.005486, mean difference in log₂(fold change) values = –0.0945, linear mixed model, two-tailed t-test on fixed effects, see Methods). h, Median fold change (log₂) in mRNA half-lives for each chromosome in Mettl3 KO over WT mESCs³⁴ (P value = 0.000225, X-chromosomal versus autosomal transcripts, mean difference in log₂-transformed fold changes = −0.22057). The absolute differences between m⁶A depletion and Mettl3 KO conditions may result from differences in the experimental setup, including the mode of Mettl3 inactivation and the method used to determine transcript half-lives.

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