Widespread occurrence of N6-methyladenosine in bacterial mRNA

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m6A as well as m6A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m6A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m6A pattern that is distinct from those in eukaryotes. Most m6A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m6A in these pathways.