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.

A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation

“…By comparing cDNA A-CLIP/IP (the A marked in red). SCARLET (site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography) is by Liu et al (2013) ) (Fig. 1D).…”

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

“…Importantly, this means that only a subset of Ψ's have been identified here. We used the evolutionary conservation of Ψ's in rRNA to validate our approach using tachyzoite rRNA; we further attempted to validate the Ψ's identified with CMCT using SCARLET (Liu et al 2013) and were able to do this for some of the highly conserved sites in the very abundant rRNA, but the amount of material we could generate for the mRNAs was not sufficient to use this method to validate Ψ's in this latter class of RNA (data not shown). Among the false negatives will be those that do not meet our Ψ criteria due to a relatively low level of pseudouridylation of a position in the total cellular pool of that transcript.…”

Genome-wide detection of high abundance N6-methyladenosine sites by microarray

“…Cumulative density curve demonstrating that meRIP-seq peaks containing the 206 SPs exhibit significantly higher enrichment scores than those from remaining probes (P < 8.06 × 10 −7 , one-sided Wilcoxon rank-sum test) using previously generated MeRIPseq data. example, using a method known as SCARLET (for, site-specific cleavage and radioactive-labeling followed by ligationassociated extraction and thin-layer chromatography) to detect m 6 A on a specific RNA, Liu et al (2013) reported that the m 6 A fraction at different sites varies from 6% to 80%. Highly enriched m 6 A sites detected in our study likely play a functional role in regulating RNA activities.…”