Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Abstract

N6-methyladenosine (m6A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m6A demethylases and cell-type and cell-state-dependent m6A patterns indicate that m6A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m6A modification include mRNA splicing, export, stability, and immune tolerance; but m6A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m6A 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 m6A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m6A modification. We applied the method to determine the m6A status at several sites in two human lncRNAs and three human mRNAs and found that m6A fraction varies between 6% and 80% among these sites. We also found that many m6A candidate sites in these RNAs are however not modified. The precise determination of m6A status in a long noncoding RNA also enables the identification of an m6A-containing RNA structural motif.