Fig. 2

The evolution of m6A sites in yeast is determined mainly by cis-elements. a Classification of the detected m6A sites in yeast into significant differentially methylated sites in cis (DM-cis) or trans (DM-trans) (absolute log2 fold change > 1.6) or into ‘invariable’ m6A sites. The left column depicts the difference in m6A levels between the two parental strains. A site labeled in red is methylated at higher levels in S. cerevisiae than in S. paradoxus, a site labeled in blue is methylated higher in S. paradoxus, whereas sites in various shades of white show little difference between the alleles. The middle column depicts the corresponding difference between the two alleles of the hybrid. The right column displays the difference between the first two columns. The color bar to the left of the heatmap indicates whether a mutation disrupting the sequence motif evolved between the two species (peach), or not (turquoise). Δparental corresponds to [log2(m6A site score S.cer)-log2(m6A site score S.par)], while ΔHybrid calculated as [log2(m6A site score S.cer allele)-log2(m6A site score S.par)]. b Differences in m6A levels across the two hybrid alleles (Y-axis) as a function of difference at corresponding sites in the parental strains (X-axis) across the 2349 identified m6A sites. c-f Sequence coverage plots of m6A-seq2 IP for S.cer, S.par, and their corresponding alleles in the hybrid. Examples of the four main types of m6A sites identified in this study are shown: DM-cis sites in which the m6A consensus motif evolved a mutation (c), DM-cis sites in which the consensus motif did not evolve a mutation (c), Invariable sites in which the m6A consensus motif did not evolve a mutation (e) and invariable sites in which the m6A consensus motif did evolve a mutation (f). Green bars indicate the position of the detected m6A and its homologous locus on the other species. The 13-nt sequence in each of the two strains/alleles is shown on the bottom, indicating the methylated adenosine (green), a motif-damaging mutation (red), or evolved m6A motif on the other species (green). g Assessment of relative essentiality of sequence composition for methylation. For each differentially methylated site harboring a mismatch between S. paradoxus and S. cerevisiae, we calculated the frequency of each mutation from any base into any other base for each of the nucleotides centered around the methylation motif, reasoning that such mutations are particularly disruptive. These frequencies were normalized by their counterparts in the m6A conserved set of sites, with the rationale that these should reflect ‘neutral’ changes with respect to m6A formation. The coefficient score is calculated as the reduction of the per-base nucleotide conservation rate in ‘differentially -methylated cis’ versus ‘invariable’ m6A sites, normalized around 0 and scaled between -1 to + 1