Fig. 3
METTL1-mediated m7G methylation is required for OCT4, Nanog, and SOX2 translation in hiPSCs. a, b Effects of METTL1 KD on stem cell marker expression. a qRT-PCR analysis assessing the mRNA levels of OCT4, Nanog, and SOX2 in control and METTL1-KD hiPSCs. b Western blot showing OCT4, Nanog, and SOX2 protein levels in control and METTL1-KD hiPSCs. c, d Effects of METTL1-KD on translational efficiency of stem cell markers. c Schematic representation of the sucrose gradient procedure followed to isolate ribosome-free and ribosome-bound RNAs. d Total and polysome-fractionated RNAs from control and METTL1-KD cells were quantified by qRT-PCR, and translational efficiency was presented as relative percent of polysome associated mRNA to input mRNA of indicated stem cell markers. e Anti-m7G Northwestern blots (upper panel). RNAs were separated on TBE-urea gels, transferred to nylon membranes, and probed with anti-m7G antibodies. Expression was compared to total RNA controls (lower panel). f Quantification of relative m7G levels. g, h Rescue of stem cell markers expression by the exogenous expression of METTL1-WT but not an enzyme-inactive mutant (Mut). g Western blot analysis of reconstituted hiPSCs. h Anti-m7G Northwestern blot of m7G modifications (upper panel) and agarose gel electrophoresis of total RNA (lower panel) in METTL1-mutant samples. i Quantification of m7G levels. j Rescue of stem cell marker expression assessed by WB analysis. Data are presented as mean ± SEM; n = 3; **P < 0.01; ***P < 0.001