Reanalyses of sequencing data by Li et al. (2017) and Safra et al. (2017) provide clear evidence of the presence of m1A in mRNA. (A) IGV views of the known m1A site at position 9 of a cytosolic and mitochondrial tRNA, showing enriched misincorporations at the first two positions of sequencing reads. (B) Barplots depicting the distribution of misincorporation events along the first 50 positions of the sequencing reads. Misincorporation in m1A9 sites (in blue) are highly skewed to occur in the first two positions, whereas misincorporation in mt-rRNA sites (in red) are relatively uniformly distributed. (C) Mismatch rates of the first five positions in sequencing reads of tRNA (left panel) and mRNA (right panel) in the input sample. (D) The mismatch rate of the first base in all reads of the “IP” and “IP + demethylation” samples, plotted along the mRNA transcripts. Upon demethylase treatment, a clear decrease of mutation rate is specifically observed for A but not T/C/G. Bin size is 10 nt. (E) Distribution of the A/C/G/T sites that pass the detection threshold by Li et al. (2017). Bin size is 10 nt. (F) A “reverse calculation” to evaluate the potential false positives caused by m1A-independent mutations. Mutation rates in the “IP” and “IP + demethylation” samples are artificially exchanged and modification calling procedure is performed using the same criteria. Very limited sites can be detected in such reverse calculation. (G) A known m1A site at position 9 in an isoform of tRNAAsp(GUC) can be identified by the end-to-end aligning mode, but not the soft clipping mode. Data taken from Li et al. (2017). (H) IGV view of an m1A site at the 5′ end of the ANKRD13A transcript. While the m1A-induced mismatch rate is decreased upon demethylase treatment (“A” site within the black lines), mutation likely arising from nontemplated additions remains the same (“C” site 5′ to the m1A site).
