Sequence and parent-of-origin dependent m⁶A contribute to allele-specific gene expression

Multiple regulatory layers influence allele-specific expression (ASE), particularly through sequence-dependent and parent-of-origin-dependent mechanisms at the transcriptional level. However, little is known about ASE regulation at the post-transcriptional level. The most prevalent post-transcriptional mRNA modification, N⁶-methyladenosine (m⁶A), plays important roles in regulating gene expression. Here, we conduct transcriptome-wide analysis of allele-specific m⁶A in mice. Using early postnatal tissues from reciprocal crosses of two divergent mouse strains, we measured allelic m⁶A differences at single-base resolution. Our study reveals widespread sequence-dependent allelic imbalance in m⁶A methylation, identifying thousands of allele-specific m⁶A (ASm⁶A) sites with statistically significant and reproducible allelic methylation differences. We find evidence of potential cis-regulatory variants within 50-nt flanking regions of ASm⁶As. Intriguingly, we detect parental effects on allelic methylation across m⁶As exhibiting parent-of-origin-dependent ASE. For both sequence- and parent-of-origin-dependent m⁶As, we observe opposing allelic preferences between methylation and expression, suggesting a potential role of ASm⁶A in regulating ASE through negative effects on gene expression. Overall, our findings reveal that both cis-acting and parent-of-origin effects influence ASm⁶A, offering new insights into post-transcriptional mechanisms of ASE regulation.