Identification of N6-methyladenosine reader proteins

Methods. 2017 Aug 15:126:105-111. doi: 10.1016/j.ymeth.2017.04.019. Epub 2017 Apr 26.

Abstract

The reversible N6-methyladenosine (m6A) modification of eukaryotic messenger RNAs (mRNAs) is a widespread regulatory mechanism that impacts every step in the mRNA life cycle. The effect of m6A on mRNA fate depends on the binding of "m6A reader" proteins - RNA binding proteins that specifically bind to RNAs containing m6A. Here, we describe an RNA pull-down method that can be used to identify novel m6A reader proteins starting from a known m6A-modified site in cellular or viral RNA. We further describe how a combination of immunoprecipitation-based sequencing methods can be used to identify m6A-modified sites bound by an m6A reader protein on a transcriptome-wide level. The discovery of new m6A reader proteins and their m6A-modified targets would provide further insight into the mechanisms and functions of m6A in the cell.

Keywords: N(6)-Methyladenosine (m(6)A); Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP); RNA binding proteins; RNA pull-down; m(6)A reader proteins; m(6)A-specific methylated RNA immunoprecipitation (MeRIP).

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / analysis
  • Adenosine / genetics
  • Adenosine / metabolism
  • Binding Sites / physiology
  • Electrophoresis, Polyacrylamide Gel / methods
  • HEK293 Cells
  • Humans
  • Protein Binding / physiology
  • RNA-Binding Proteins / analysis
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism*

Substances

  • RNA-Binding Proteins
  • N-methyladenosine
  • Adenosine