NAD+-capped RNAs are preferentially produced from inducible genes in response to growth conditions in E. coli

Recent findings regarding NAD+-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ non-canonical RNA capping to regulate genes, a previously unrecognized mechanism. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition click chemistry reaction to label NAD-RNAs. However, copper can fragment RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition for labeling NAD-RNAs, followed by identification of tagged RNA by direct RNA sequencing. Using this method, we compared NAD-RNA and total transcript profiles of E. coli cells in the exponential and stationary phases and identified hundreds of NAD-RNA species. For some genes, the majority of their transcripts were found as NAD-RNAs. Our study indicates that NAD-RNAs are preferentially produced from inducible genes in response to different growth conditions. Overall design: Four groups of Samples, exponential and stationary plus and minus