Supplementary Figure 4: m6A modification regulates mRNA stability.

A. Enrichment of up- or down-regulated Mettl3 and Mettl14 targets in kd versus control cells. Arrows indicate up- or down regulation of gene expression. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA against Mettl14. B. Comparison of cumulative transcript abundance in cells treated with Actinomycin D for 0, 4, and or 8 hrs by microarray analysis. RNAs from 0 hr serves as base-line. KS test, p<2.2e−16 comparing the changes in relative RNA levels from 4 to 8 hrs between Mettl targets and non-targets in both Mettl3 kd and Mettl14 kd cells. C. Enrichment of bivalent and pluripotency (Oct4 PPI) genes in shared Mettl3 and Mettl14 targets. D. meRIP-qPCR of specific bivalent and pluripotency genes (grey area). E. Enrichment of Mettl3 and Mettl14 targets showing increased RNA stability in kd cells among bivalent and pluripotency genes. F. Measurement of a percentage of m6A/A ratio by MS in undifferentiated (day 0), day6, and day 12 differentiated mESCs. G. RT-qPCR of Mettl3, Mettl14, and Rex1 expression in undifferentiated and differentiated mESCs. H. meRIP-qPCR of specific bivalent (gray area) and pluripotency genes in mESCs during differentiation. Error bars from panels D and G-H represent means ± SEM from 3 separate experiments. One-tailed Student’s t-test, *P<0.05,**P<0.01,***P<0.001,****P<0.0001 vs. scramble control. Scr: scramble.