Deciphering the "m 6 A Code" via Antibody-Independent Quantitative Profiling

Cell. 2019 Jul 25;178(3):731-747.e16. doi: 10.1016/j.cell.2019.06.013. Epub 2019 Jun 27.

Abstract

N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / analysis
  • Adenosine / immunology
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO / genetics
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO / metabolism
  • Animals
  • Antibodies / immunology
  • Chromatography, High Pressure Liquid
  • Embryoid Bodies / metabolism
  • Embryonic Stem Cells
  • Endoribonucleases / metabolism
  • Humans
  • Meiosis
  • Methylation
  • Mice
  • Nucleotide Motifs
  • RNA, Messenger / chemistry*
  • RNA, Messenger / metabolism
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, RNA / methods*
  • Tandem Mass Spectrometry

Substances

  • Antibodies
  • RNA, Messenger
  • N6-methyladenosine (m6A)
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO
  • FTO protein, human
  • Endoribonucleases
  • Adenosine