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Mapping N 6 ‐Methyladenosine (m 6 A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches
Author(s) -
Hartstock Katja,
Rentmeister Andrea
Publication year - 2019
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201804043
Subject(s) - rna , computational biology , immunoprecipitation , messenger rna , derivatization , enzyme , n6 methyladenosine , chemistry , biology , biochemistry , mass spectrometry , gene , chromatography , methyltransferase , methylation
N 6 ‐Methyladenosine (m 6 A) is the most abundant internal modification in eukaryotic mRNA. Specific m 6 A 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 m 6 A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m 6 A mapping. Starting with the seminal m 6 A‐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 m 6 A‐sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m 6 A. Finally, single‐molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.