Mapping N⁶‐Methyladenosine (m⁶A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches

Abstract

N6‐Methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA. Specific m6A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m6A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m6A mapping. Starting with the seminal m6A‐sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo‐cross‐linking and remaining challenges. As an alternative, antibody‐free approaches will be presented. These include wild‐type or engineered m6A‐sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m6A. Finally, single‐molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.

Sequence-specific m6A demethylation in RNA by FTO fused to RCas9

“…The method could even be used to study other functions of FTO in a sequence-specific manner, e.g., specific demethylation of m 1 A in tRNA, which was recently identified to be demethylated by FTO (Wei et al 2018). The analysis of off-target effects in cells is demanding and in this case could be performed by transcriptome-wide m 6 A-detection methods (Dominissini et al 2012;Meyer et al 2012;Schwartz et al 2013;Chen et al 2015;Ke et al 2015;Linder et al 2015;Molinie et al 2016;Hartstock and Rentmeister 2018). Since it is hard to obtain information about the stoichiometry of m 6 A at a certain position, this could be backed up by testing the m 6 Alevels in mRNA by LC-MS/MS.…”

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…A number of recent reviews and surveys describe the use of biorthogonal metabolic labeling for the detection of RNA and RNA modifications [5,[39][40][41].…”

“…The conversion of C-to-U residues in DNA and RNA by bisulfite is probably the most used chemical reaction in nucleic acid chemistry. Bisulfite RNA sequencing, which was adapted from 5mC detection in DNA, was described for m 5 C mapping and quantification by Schaefer and Lyko [47]. The method is generally considered to be relatively robust for abundant RNAs, such as tRNA and rRNA [48][49][50][51], but the reliability of the data obtained on less abundant RNAs, including mRNAs and lncRNAs [52][53][54][55], is still being debated [56][57][58].…”

“…In the context of the COVID-19 pandemic, the direct RNA nanopore sequencing of full-length coronavirus genomic RNA allowed for us to predict multiple sites of m 5 C modification in SARS-Cov-2 [161]. However, the existence of these modifications in SARS-Cov-2 is still controversial, since another study utilizing nanopore sequencing with more rigorous controls did not confirm their presence [162].…”

“…The analysis of RNA modifications by NGS is still a very recent topic (the first publications in the field are from 2012); however, a number of excellent review articles have already covered previous achievements [2][3][4][5][6][7]. In this comprehensive review, we will focus on the most recent and emerging approaches that have appeared during the last 2-3 years, and thye are not yet fully included in the relevant review literature.…”