N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100–200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here, we describe a method for using anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Then, we describe how to use these mutational signatures to map m6A residues at nucleotide resolution. Taken together, our protocol allows for high-throughput detection of individual m6A residues throughout the transcriptome.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
We thank members of the Jaffrey lab for their helpful comments and support. This work was supported by NIH grants NIDA DA037150 (S.R.J.), T32 CA062948 (A.O.-G.), and a German Research Foundation (DFG) fellowship (B.L.).
Dominissini D, Moshitch-Moshkovitz S, Schwartz S et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206CrossRefPubMedGoogle Scholar
Perry R, Kelley D, Friderici K et al (1975) The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5′ terminus. Cell 4:387–394CrossRefPubMedGoogle Scholar
Desrosiers R, Friderici K, Rottman F (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A 71:3971–3975CrossRefPubMedPubMedCentralGoogle Scholar
Schibler U, Kelley D, Perry R (1977) Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. J Mol Biol 115:695–714CrossRefPubMedGoogle Scholar
Schwartz S, Mumbach M, Jovanovic M et al (2014) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep 8:294–296Google Scholar
Webb S, Hector R, Kudla G et al (2014) PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast. Genome Biol 15:R8CrossRefPubMedPubMedCentralGoogle Scholar