MePMe-seq: Antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing

Abstract Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Current insights rely on the ability to make a modified nucleoside amenable to sequencing. Most of the modifications are methylations involving the co-factor S -adenosyl-L-methionine (SAM), however, simultaneous detection of different methylation sites in the same sample has remained elusive. We present metabolic labeling with propargyl-selenohomocysteine (PSH) in combination with click chemistry to detect N 6 - methyladenosine (m 6 A) and 5-methylcytidine (m 5 C) sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). Intracellular formation of the corresponding SAM analogue leads to detectable levels of N 6 -propargyl-A (prop 6 A) and 5-propargyl-C (prop 5 C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m 6 A and m 5 C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.