MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing
Katja Hartstock, Nadine A. Kueck, Petr Špaček, Anna Ovcharenko, Sabine Hüwel, Nicolas V. Cornelissen, Amarnath Bollu, Christoph Dieterich, Andrea Rentmeister
Abstract
Abstract Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S -adenosyl- L -methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N 6 -methyladenosine (m 6 A) and 5-methylcytidine (m 5 C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m 6 A from 2′- O -methyladenosine (A m ) and N 1-methyladenosine (m 1 A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.