Probing N super(6)-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

• Published in: RNA (Cambridge) 2013-12, Vol.19 (12), p.1848-1856
• Main Authors: Liu, M, Parisien, M, Dai, Q, Zheng, G, He, C, Pan, T
• Format: Article
• Language: English
• ISSN: 1355-8382

N super(6)-methyladenosine (m super(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (IncRNA). Recent discoveries of two m super(6)A demethylases and cell-type and cell-state-dependent m super(6)A patterns indicate that m super(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m super(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m super(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m super(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m super(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m super(6)A modification. We applied the method to determine the m super(6)A status at several sites in two human IncRNAs and three human mRNAs and found that m super(6)A fraction varies between 6% and 80% among these sites. We also found that many m super(6)A candidate sites in these RNAs are however not modified. The precise determination of m super(6)A status in a long noncoding RNA also enables the identification of an m super(6)A-containing RNA structural motif.