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 N6-methyladenosine (m6A) and 5-methylcytidine (m5C) 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 N6-propargyl-A (prop6A) and 5-propargyl-C (prop5C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m6A and m5C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.

RNA methylation in plants: An overview

“…In this method, metabolic labeling with propargylselenohomocysteine in along with click chemistry is used to detect N6A and m5C sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation (Hartstock et al, 2023). In this method first, Metabolic labeling of cells with propargyl-selenohomocysteine is performed.…”

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

“…propargyl residues, has enabled their incorporation into RNA in lieu of methyl groups. Subsequent derivatization by click chemistry was exploited for determination of modification sites ( 75 , 76 , 66 ).…”

DNA‐Methyltransferasen und AdoMet‐Analoga als Werkzeuge für die Molekularbiologie und Biotechnologie

“…Da die Propargyl‐Gruppe das kleinste terminale Alkin für MTase‐basierte Markierung darstellt und mit anschließender Klick‐Chemie weiter funktionalisiert werden kann, ist SeAdoYn von besonderer Bedeutung. Trotz vielversprechender Beispiele limitiert die aufwändigere Synthese von SeAdoMet‐Analoga eine breitere Anwendung 43–46.…”