Use of NAD-Seq to Profile NAD+-Capped RNAs in Plants.

Abstract

A 7-methylguanosine (m7G) cap in the 5′ end of eukaryotic mRNA transcript to protect mRNAs from degradation and regulate gene expression has been well characterized. Recently, some RNAs were found to contain a NAD+ cap, which is a different type of 5′ cap structure in eukaryotic organisms, including bacteria, yeast, mammalian cells, and Arabidopsis thaliana (arabidopsis) plant. NAD captureSeq was the first method to label NAD+-capped RNAs (NAD-RNAs) and uses ADP ribose cyclase (ADPRC)-catalyzed replacement of the nicotinamide of NAD+ with an alkynyl alcohol followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to biotinylate NAD-RNA. In the next step, the eluted RNAs are subjected to library preparation and sequencing, followed by determination of NAD+-capped RNA in the transcriptome. Improved methods are emerging to determine NAD-RNAs, such as NAD tagSeq and strain-promoted azide–alkyne cycloaddition reaction (SPAAC)-NAD-Seq. Commonly, those methods use an enzymatic reaction followed by a click chemistry reaction to label NAD-RNAs with biotin or synthetic RNA tag (TagRNA).