Fig. 5

Sequence and RNA secondary structure around methylation consensus motifs determine variability in m6A levels across mammalian individuals of the same species. a Scheme depicting the design of a massive parallel reporter assay aiming to establish the impact of secondary structure on m6A formation. For each of 120 m6A sites, we designed a set of oligos in which we systematically shifted the relative position of the methylated DRACH motif from a fully open loop (methylated adenosine at position 56) into the stem (starting from position 62). b Distribution of m6A enrichment (IP/input) levels as a function of the relative position along the sequence (and secondary structure), depicting a gradual decrease in m6A levels as the consensus motif is shifted from positions in the loop to positions in the stem. c Boxplot displaying the differences in the predicted MFE for sites detected either exclusively in the castaneous strain, only in house mice, or in both, as in Fig. 4h (n = 969). d Left: distribution of beta values, obtained from Zhang et al. (22), across 101 ‘proximal’ m6A-QTLs, binned based on whether the m6A QTLs lead to the formation of a site exclusively at the reference allele, alternative allele or does not impact methylation status. The beta represents the correlation between the SNP and allele-specific methylation scores of 60 YRI human individuals, with negative beta indicating that the hg19 reference genome variant is methylated, while positive beta indicates that the alternative variant leads to higher methylation levels at the locus. Right: for the same 97 m6A sites, displayed are the m6A scores ratio for the synthesized 101-nt window sequence centered around the methylated adenosine, derived from either the reference or the alternative sequence. e Boxplot as in (c) for 60 different human individuals as published in Zhang et al. Methylation sites exclusive to the reference allele were defined as ones with beta > 1, whereas ones exclusive to the alternative allele were defined as beta < -1. Betas within the range of -1 to 1 were considered to display similar methylation patterns between the published genetic variants. Only sites without motif-damaging SNP and at least one allele with a predicted solid structure (MFE < -8 kcal/mol) are displayed (n = 152). f An example of an m6A-QTL site that was found by Zhang et al. (22), which did not harbor an m6A consensus motif disrupting SNP. The top panel is a boxplot representing the m6A scores for each of the population genotypes (n = 60 independent samples). Boxplots correspond to the median, Q1 and Q3, whiskers mark Q1-1.5 IQR and Q3 + 1.5 IQR. The lower panel illustrates the predicted MFE secondary structure around the DRACH motif nearest to the m6A-QTL. The m6A-QTL sequence variation is marked in blue on the reference genome and in red in the alternative allele