N6-methyladenosine (m⁶A) is the most common internal mRNA modification in eukaryotes, affecting various stages of RNA processing and metabolism. To examine its evolutionary conservation and functional importance, we analysed direct RNA nanopore sequencing data of six tissues from six vertebrate species, comprising five mammals and one bird species (as an outgroup). Our research revealed that over 38% of human genes include at least one conserved m⁶A site, primarily located near stop codons and within 3′ untranslated regions (3′UTRs). We found that the canonical DRACH motif accounted for a majority of conserved m6A sites, with 80% conserved across two or more species. Nevertheless, non-DRACH and AG-rich motifs, despite their lower frequency, demonstrated notable conservation, suggesting possible regulatory functions that might be tissue-specific.
To further understand the functional importance of these conserved m⁶A sites, we incorporated single-nucleotide polymorphism (SNP) data from the ClinVar and dbSNP databases. Interestingly, while we found a significant depletion of m6A-SNPs compared to a control set, we observed an enrichment of variants of unknown significance (VUS) associated with m6A. Gene set enrichment analysis (GSEA) of genes containing these m6A-SNPs in brain tissues revealed relationships with brain development and function. Moreover, the overlap of SNPs with expression quantitative trait loci (eQTL) data revealed that SNPs disrupting m⁶A motifs might be highly associated with differential gene expression profiles, highlighting the regulatory influence of m⁶A modifications.
Altogether, our analysis highlights the evolutionary conservation of mRNA m⁶A modifications and their functional significance in gene regulation. Thus, enhancing our understanding of m⁶A function in health and disease.