Extended Data Fig. 6: Screening for candidate anti-pausing factors.
From: m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency
a. Quantification of the number of expressed genes in Mettl3+/+ and Mettl3−/− ESCs based on intronic RNA-seq, in control and paused conditions. Expressed genes are further defined as having high expression (log2 normalized reads > 5, n = 3 biological replicates). b. Heatmap of gene expression based on intronic reads for all genes expressed in Mettl3+/+ or Mettl3−/− ESCs (left) with average expression per sample (right, scored as median z-scores of all genes), showing defective hypotranscription in paused Mettl3−/− ESCs. c. Identification of putative anti-pausing factors kept in check by m6A methylation and thereby destabilization of their transcript in paused pluripotency, based on RNA-seq, MeRIP-seq and SLAM-seq data in ESCs (see Methods for details). d. Expression levels (log2 cpm) of the Myc factors in diapaused embryos (left, data from Boroviak et al.) and paused ESCs (right). Horizontal bars represent the mean, with 3 biological replicates per group, except for diapaused embryos which has 2 replicates. e. mTOR inhibition by dual knockdown of Rptor and Rictor reduces Mycn expression measured by RT-qPCR in ESCs in FBS/LIF/2i medium (n = 4 biological replicates). Data are mean ± SD (a, b, e). P-values (as indicated on figure) by two-way ANOVA with Tukey’s multiple comparisons test (a), two-tailed Student’s t-tests (b), and one-way ANOVA with Dunnett’s multiple comparison tests (e).
