Extended Data Fig. 2: Paused Mettl3−/− ESCs acquire a distinct gene expression profile.
From: m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency
a. Quantification of total RNA per cell in Mettl3+/+ #2 and Mettl3−/− #2–4 ESCs, in control and paused conditions. Data are mean ± SD, n = 5 biological replicates. b. Decrease in nascent RNA per cell, as measured by EU incorporation with nuclear signal quantification, in wildtype ex vivo paused or hormonally diapaused blastocysts, compared to control E3.5 embryos. Number of embryos (n) as indicated. Scale bars = 30 µm. c. Strategy for RNA-seq with cell number-normalization using ERCC spike-in RNAs. d. Quantification of the number of expressed genes in Mettl3+/+ and Mettl3−/− ESCs, in control and paused conditions. Expressed genes are further defined as having high expression (log2 normalized reads > 5, n = 3 biological replicates). e. Number of differentially expressed genes (fold-change > 1.5 and adjusted P < 0.05) upon pausing, in Mettl3+/+ and Mettl3−/− ESCs. f. PCA plot for all expressed genes across all samples, showing across PC1 that Mettl3+/+ ESCs acquire a more divergent expression profile upon pausing than Mettl3−/− ESCs, relative to respective control condition. g-h. Gene expression changes (log2 fold-changes) of gene sets selected from the ‘GO biological processes’ (g) and ‘hallmarks’ (h) collections, showing incomplete downregulation in paused Mettl3−/− ESCs. i. Western blot of total and phosphorylated mTOR, and of the downstream targets of mTORC1 (Ulk1, 4Ebp1 and S6K1) (left, representative of 4 biological replicates). Quantification of phosphorylated levels, normalized to total levels, show no significant change in paused ESCs between Mettl3+/+ and Mettl3−/− (right). Data are mean ± SD (b, d, g-i). P-values (as indicated on figure) by two-way ANOVA with Dunnett’s multiple comparison tests (a, i), one-way ANOVA with Dunnett’s multiple comparison tests (b), two-tailed unpaired Student’s t-tests (d, g-h).
