Extended Data Fig. 3: METTL3 catalytic activity is required for acceleration of Xist-mediated silencing.
From: m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics
a, Schematic showing the strategy for transgene complementation. METTL3 transgene is inserted into Rosa26 locus and transcribed under the native Rosa26 promoter. b, Western blots showing the protein level for GFP-METTL3D395A (left: 5B and 9F are two independent clones) and GFP-METTL3 (right: 1A and 10C are two independent clones), along with METTL3-FKBP12F36V and METTL14, after 2 or 24 h dTAG-13 treatment. The top and second from top gels are short and long exposure respectively. TBP and SETDB1 loading controls. Note that clones 5B, 9F, 1A, and 10C are Xcast0 due to loss of X129. c, Western blots showing the protein level for GFP-METTL3D395A, along with METTL3-FKBP12F36V and METTL14, after 2 or 24 h dTAG-13 treatment. The bottom and second from bottom gels are short and long exposure respectively. SETDB1 loading controls. Note that clone 1F has retained both X129 and Xcast. Here Xcast is the Xi chromosome. d, Barplot showing Mettl3 transcript level based on ChrRNA-seq data for cells harbouring GFP-METTL3 (represented by clone 1A) or GFP-METTL3D395A (represented by clone 9F). Two replicates for each clone are included. Expression level is the sum of reads from native Mettl3 locus and transgenic Rosa26 locus. The yellow bars represent the level from cells expressing the transgene encoding METTL3D395A. Each bar represents one sample. e, Boxplot showing average gene expression levels for X-linked genes in control or dTAG-13 treated samples. The averages are calculated from two clones shown in (b). Yellow boxplots represent the level from cells expressing the transgene encoding METTL3D395A. Significance was determined by use of a two-tailed Wilcoxon test. Centre lines indicate the median, box limits indicate the first and third quartiles, and whiskers indicate 1.5× IQR. f, Xist expression level from samples of GFP-METTL3 or GFP-METTL3D395A clones. Clone names are indicated as above. Y-axis denotes the RPM value, as shown in Fig. 2a. Yellow barplots represent the level from cells expressing the transgene encoding METTL3D395A. Each bar represents one sample.
