Fig 2: FTO overexpression causes aberrant mRNA methylation-suppression in the nucleus. a Immunofluorescence images of WT versus Fto-KO cells. Anti-FTO i and anti-FTO ii antibodies are respectively Abcam ab126605 and Santa Cruz sc271713. Anti-FTO i is specific for endogenous FTO while anti-FTO ii is not. Scale bar denotes 10 µm. b Immunofluorescence images of C-terminal-3 × -FLAG-tagged-WT-FTO in HEK293T. Scale bar denotes 10 µm. c, d m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to representative mRNAs. Sequence corresponds to the same strand as the m6A site. Blue horizontal bars represent transcript models. e Scatterplot of average RML of WT versus WT-Fto-OE cells. WT-FTO-affected are sites with RML reduction of at least log2fold of 2 (Student’s t-test p < 0.05) in WT-Fto-OE compared to WT cells. f MEME analysis of the sequence contexts of FTO-affected sites. g Model for cytoplasmic RNA methylation-reversal. h Model for disruptive methylation suppression by RNA demethylases. (Left panel) Most methylated RNAs (blue) are not demethylated by demethylases but simply undergo eventual RNA decay. (Middle panel) Selected RNAs (green) that ought to remain unmethylated are acted on almost simultaneously by both methyltransferase and demethylase, resulting in no net accumulation of RNA methylation. (Right panel) In the absence of demethylases, RNA methylation accumulates anomalously, which disrupts regular RNA processing.

Fig 3: METTL16 depletion reduces methylation of a plethora of m6A sites beyond its direct UACAGAGAA targets. a m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to the Mat2a 3'UTR. Locations of ‘UACAGAGAA’ and similar motifs are denoted by roman numerals. Sequence corresponds to the same strand as the m6A site. Blue horizontal bars represent transcript models. b Magnified representations of the Mat2a 3'UTR at positions i, ii and iii as denoted in a. c Scatterplot of average RML of WT versus Mettl16-KD cells. METTL16-dependent are sites with RML reduction of at least log2fold of 2 (Student’s t-test p < 0.05) in Mettl16-KD compared to WT cells. d Metagene distribution profile of METTL16-dependent m6A/m6Am. e MEME analysis of the sequence context of METTL16-dependent m6A/m6Am. f, g Venn diagram representing overlap between METTL16-dependent with METTL3-dependent (f) or PCIF1-dependent (g) sites. p-value of overlap by chance was calculated with a hypergeometric test.

Fig 4: FTO loss causes m6Am accumulation that disrupts binding of specific snRNA precursors to nuclear export machinery. a Scatterplot of average RML of WT versus Fto-KO cells. FTO-regulated (‘X’s) are sites with RML accumulation of at least log2fold of 1 (Student’s t-test p < 0.05) in Fto-KO compared to WT cells. b, c m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to the 5' ends of U1 snRNA (b) and U4 snRNA (c). FTO-regulated m6Am sites are denoted by green dots. Sequence corresponds to the same strand as the m6A/m6Am sites. Blue horizontal bars represent transcript models. d Box and whisker plots of m6Am/(m6Am + Am) percentages in WT (blue) and Fto-KO (orange) cells as measured by UHPLC-MS/MS. Represented are median and interquartile ranges for 3 biological replicates. # and ## denote p < 0.05 and 0.005 respectively (Student’s t-test). Values for WT cells are in the non-quantifiable range. e Box and whisker plots of ratio of sRNA levels (relative to 7SL scRNA) in nucleus over cytoplasm in WT (blue) and Fto-KO (orange) cells. Represented are median and interquartile ranges for 6 biological replicates, normalised to the corresponding median values in WT cells. A higher ratio corresponds to the sRNA localising more to the nucleus than the cytoplasm. f NCBP2-RIP analysis of WT (blue) and Fto-KO (orange) lysates. Relative NCBP2 enrichment is the ratio of each sRNA pulled down with anti-NCBP2 over non-specific IgG, normalised to housekeeping Gapdh mRNA. Represented are box and whisker plots with median and interquartile ranges for at least three biological replicates, normalised to the corresponding median values in WT cells. g ROC curve for FTO regulation as a predictor of steady-state non-methylation in WT cells. Each curve represents FTO-regulated sites as defined by a different minimum log2FC RML accumulation cutoff (Student’s t-test p < 0.05) in Fto-KO over WT cells. AUC values for each log2FC are also provided. For box and whisker plots, centre line, box boundaries, lower whisker, upper whisker and empty circles represent the median, interquartile range, minimum value, maximum value and outliers respectively. n.s., * and ** respectively denote not significant, p < 0.1 and p < 0.01 (Student’s t-test).

Fig 5: m6ACE-seq quantitatively maps RML reductions at individual PCIF1-dependent m6Am sites. a m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to a representative gene (center). PCIF1-dependent and METTL3-dependent sites are respectively denoted by red angles and red dots. Sequence corresponds to the same strand as the m6A/m6Am sites. Blue horizontal bars represent transcript models. Magnified views of the 5'UTR (left) and 3'UTR (right) are also displayed. b Scatterplot of average RML of WT versus Pcif1-KO cells. PCIF1-dependent are sites with RML reduction of at least log2fold of 2 (Student’s t-test p < 0.05) in Pcif1-KO compared to WT cells. c Functional annotation enrichment of genes containing PCIF1-dependent m6Am with significance of enrichment denoted as -log10FDR. d Metagene distribution profile of PCIF1-dependent m6Am. e MEME analysis of the sequence context of PCIF1-dependent m6Am. f Venn diagram representing overlap between 5'UTRs containing PCIF1-dependent m6Am with 5'UTRs containing PCIF1-independent sites. p-value of overlap by chance was calculated with a hypergeometric test. PCIF1-independent sites are sites where average RML reduction from WT to PCIF-KO is not significantly more than 1.5 fold (Student’s t-test p < 0.05). g m6ACE (green) overlaid on Input (orange) read-start RPM counts mapped to a representative 5'UTR. PCIF1-dependent and PCIF1-independent sites are respectively denoted by red angles and black dots. Sequence corresponds to the same strand as the m6A/m6Am sites. Blue horizontal bars represent transcript models. h Pie charts representing alignment of all identified m6A/m6Am (top) or PCIF1-dependent m6Am (bottom) with respect to CAGE-seq annotated TSSs39.