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

Abstract

N6-methyladenosine (m 6 A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m 6 A demethylases and cell-type and cell-state-dependent m 6 A patterns indicate that m 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 6 A modification include mRNA splicing, export, stability, and immune tolerance; but m 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 6 A status at any site in mRNA/lncRNA, termed s ite-specific c leavage a nd r adioactive-labeling followed by l igation-assisted e xtraction and t hin-layer chromatography (SCARLET). The method determines the precise location of the m 6 A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m 6 A modification. We applied the method to determine the m 6 A status at several sites in two human lncRNAs and three human mRNAs and found that m 6 A fraction varies between 6% and 80% among these sites. We also found that many m 6 A candidate sites in these RNAs are however not modified. The precise determination of m 6 A status in a long noncoding RNA also enables the identification of an m 6 A-containing RNA structural motif.