Journal
METHODS
Volume 126, Issue -, Pages 105-111Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2017.04.019
Keywords
N-6-Methyladenosine (m(6)A); RNA binding proteins; m(6)A reader proteins; RNA pull-down; Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP); m(6)A-specific methylated RNA immunoprecipitation (MeRIP)
Funding
- National Institutes of Health [R01GM113194, F30GM120917]
- National Institutes of Health Medical Scientist Training Program [T32GM007281]
- University of Chicago Biological Sciences Division and Frank Family Endowment
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The reversible N-6-methyladenosine (m(6)A) modification of eukaryotic messenger RNAs (mRNAs) is a widespread regulatory mechanism that impacts every step in the mRNA life cycle. The effect of m(6)A on mRNA fate depends on the binding of m(6)A reader proteins - RNA binding proteins that specifically bind to RNA5 containing m(6)A. Here, we describe an RNA pull-down method that can be used to identify novel m(6)A reader proteins starting from a known m(6)A-modified site in cellular or viral RNA. We further describe how a combination of immunoprecipitation-based sequencing methods can be used to identify m(6)A-modified sites bound by an m(6)A reader protein on a transcriptome-wide level. The discovery of new m(6)A reader proteins and their m(6)A-modified targets would provide further insight into the mechanisms and functions of m(6)A in the cell. (C) 2017 Elsevier Inc. All rights reserved.
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