论文信息 - Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).

Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).

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.

[1] R. Desrosiers,et al. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[2] Grzegorz Kudla,et al. PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast , 2014, Genome Biology.

[3] Schraga Schwartz,et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. , 2014, Cell reports.

[4] Christopher E. Mason,et al. Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome , 2015, Nature Methods.

[5] Robert P. Perry,et al. The methylated constituents of L cell messenger RNA: Evidence for an unusual cluster at the 5′ terminus , 1975, Cell.

[6] O. Elemento,et al. Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons , 2012, Cell.

[7] Aaron R. Quinlan,et al. BIOINFORMATICS APPLICATIONS NOTE , 2022 .

[8] U. Schibler,et al. Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. , 1977, Journal of molecular biology.

[9] Julian König,et al. Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions , 2012, Genome Biology.

[10] M. Kupiec,et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.