Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

Abstract

N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reactions cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the birds-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/460679v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@15845aeorg.highwire.dtl.DTLVardef@46a95aorg.highwire.dtl.DTLVardef@118633dorg.highwire.dtl.DTLVardef@1b52f9_HPS_FORMAT_FIGEXP M_FIG C_FIG