Genome-wide identification of mRNA 5-methylcytosine in mammals

Accurate and systematic transcriptome-wide detection of 5-methylcytosine (m⁵C) has proved challenging, and there are conflicting views about the prevalence of this modification in mRNAs. Here we report an experimental and computational framework that robustly identified mRNA m⁵C sites and determined sequence motifs and structural features associated with the modification using a set of high-confidence sites. We developed a quantitative atlas of RNA m⁵C sites in human and mouse tissues based on our framework. In a given tissue, we typically identified several hundred exonic m⁵C sites. About 62-70% of the sites had low methylation levels (<20% methylation), while 8-10% of the sites were moderately or highly methylated (>40% methylation). Cross-species analysis revealed that species, rather than tissue type, was the primary determinant of methylation levels, indicating strong cis-directed regulation of RNA methylation. Combined, these data provide a valuable resource for identifying the regulation and functions of RNA methylation.