Comprehensive Analysis of mRNA Methylation Reveals Pervasive Adenosine Methylation in 3' UTRs

Epigenetics is widely studied in DNA but was only recently discovered in RNA, where FTO, an obesity risk gene, implicates the methylation of the N⁶ position of adenosine (m⁶A) of post-transcriptional RNA in energy homeostasis. We devised a novel immunoprecipitation (IP) protocol, MeRIP-seq, to isolate mRNA for m⁶A sites, coupled with a novel computational peak-finder, MeRIPPeR, to localize these sites throughout the genome. MeRIP-seq isolates fragmented and RiboMinus-treated mRNA using an antibody specific for m⁶A. The IP fragments are sequenced using next-generation sequencing (NGS). MeRIPPeR uses Fishers’ Exact Test as its primary peak-finding metric to localize m⁶A sites throughout the genome. These sites are then analyzed using known RefSeq gene annotations. Using MeRIP-seq and MeRIPPeR, we identified mRNAS of 7,676 genes which contained m⁶A and discovered that m⁶A sites are especially enriched near stop codons and in the 5’ end of the 3’ UTR. We confirmed some m⁶A sites using biotinylated DNA probes and streptavidin Dynabeads, which demonstrates that MeRIP-seq successfully isolates mRNAs with m⁶A sites. Our results indicate that m⁶A is a common modification of mRNA and show insight into the role of RNA epigenetics.