Abstract
N 6-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 via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Linder B, Grozhik A, Olarerin-George A et al (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767–772
Meyer K, Saletore Y, Zumbo P et al (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′UTRs and near stop codons. Cell 149:1635–1646
Dominissini D, Moshitch-Moshkovitz S, Schwartz S et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206
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 4:387–394
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 71:3971–3975
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 115:695–714
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 8:294–296
Sugimoto Y, Konig J, Hussain S et al (2012) Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions. Genome Biol 13:R67
Dodt M, Roehr J, Ahmed R et al (2012) FLEXBAR—flexible barcode and adapter processing for next-generation sequencing platforms. Biology 1:895–905
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:R8
Quinlan A, Hall I (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842
Acknowledgments
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.).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Grozhik, A.V., Linder, B., Olarerin-George, A.O., Jaffrey, S.R. (2017). Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP). In: Lusser, A. (eds) RNA Methylation. Methods in Molecular Biology, vol 1562. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6807-7_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6807-7_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6805-3
Online ISBN: 978-1-4939-6807-7
eBook Packages: Springer Protocols