title: Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.
abstract: N(6)-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 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(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.
authors.forename: Nian
authors.initials: N
authors.lastname: Liu
authors.forename: Marc
authors.initials: M
authors.lastname: Parisien
authors.forename: Qing
authors.initials: Q
authors.lastname: Dai
authors.forename: Guanqun
authors.initials: G
authors.lastname: Zheng
authors.forename: Chuan
authors.initials: C
authors.lastname: He
authors.forename: Tao
authors.initials: T
authors.lastname: Pan
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C557041:MALAT1 long non-coding RNA, human; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; C010223:N-methyladenosine; D000241:Adenosine
keywords: N6-methyladenosine; RNA modification; single nucleotide resolution