NAD tagSeq reveals that NAD+-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis

The 5′ end of a eukaryotic messenger RNA generally contains an 7-methylguanosine (m⁷G) cap, which has an essential role in regulating gene expression. Recent discoveries of RNAs with a noncanonical NAD⁺ moiety indicate the existence of a previously unknown mechanism for controlling gene expression. We have developed a method termed NAD tagSeq for the accurate identification and quantification of NAD⁺-capped RNAs and for revealing the complete sequences of NAD-RNAs using single-molecule RNA sequencing. Using this method, we found that NAD-RNAs in Arabidopsis were mostly derived from protein-coding genes and that they have essentially the same overall sequence structures as the canonical m⁷G-RNAs. The identification of NAD-RNAs and their sequence structures facilitates the elucidation of their possible molecular and physiological functions.