Probing RNA Modification Status at Single-Nucleotide Resolution in Total RNA.

RNA modifications, with over one hundred known so far, are commonly proposed to fine-tune the structure and function of RNA. While modifications in rRNA and tRNA are used to modulate RNA folding and decoding properties, little is known about the function of internal modifications in mRNA/lncRNA, which includes N(6)-methyl adenosine (m(6)A), 5-methyl cytosine (m(5)C), 2'-O-methylated nucleotides (Nm), pseudouridine (Ψ), and possible others. Functional studies of mRNA/lncRNA modifications have been hindered by the lack of methods for their identification at single-nucleotide resolution. Challenges for the determination of mRNA/lncRNA modifications at single-nucleotide resolution are mainly due to the low abundance of mRNA/lncRNA. Traditional deep sequencing methods cannot identify mRNA/lncRNA modifications, such as m(6)A, m(5)C, Nm, and Ψ, because reverse transcriptase is insensitive to their presence in cDNA synthesis. Antibody-based approach enables the identification of m(6)A regions in mRNA/lncRNA, but currently at ~100 nucleotide resolution. Here, we describe a method that accurately identifies m(6)A position and modification fraction in human mRNA and lncRNAs at single-nucleotide resolution, termed "Site-specific Cleavage And Radioactive-labeling followed by Ligation-assisted Extraction and Thin-layer chromatography (SCARLET)." This method combines two previously established techniques, site-specific cleavage and splint ligation, to probe the RNA modification status at any mRNA/lncRNA site in the total RNA pool. SCARLET can potentially analyze any nucleotide that maintains Watson-Crick base pairing in the transcriptome and determine whether it contains m(6)A, m(5)C, Nm, Ψ, or other modifications yet to be discovered. Precise determination of the position and modification fraction of RNA modifications reveals crucial parameters for functional investigation of RNA modifications.