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 mRNA transcript generally has a 7-methylguanosine (m⁷G) cap that protects mRNA from degradation and mediates almost all other aspects of gene expression. Some RNAs in Escherichia coli, yeast, and mammals were recently found to contain an NAD⁺ cap. Here, we report the development of the method NAD tagSeq for transcriptome-wide identification and quantification of NAD⁺-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq can allow more accurate identification and quantification of NAD-RNAs, as well as reveal the sequences of whole NAD-RNA transcripts using single-molecule RNA sequencing. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis were produced by at least several thousand genes, most of which are protein-coding genes, with the majority of these transcripts coming from <200 genes. For some Arabidopsis genes, over 5% of their transcripts were NAD capped. Gene ontology terms overrepresented in the 2,000 genes that produced the highest numbers of NAD-RNAs are related to photosynthesis, protein synthesis, and responses to cytokinin and stresses. The NAD-RNAs in Arabidopsis generally have the same overall sequence structures as the canonical m⁷G-capped mRNAs, although most of them appear to have a shorter 5′ untranslated region (5′ UTR). The identification and quantification of NAD-RNAs and revelation of their sequence features can provide essential steps toward understanding the functions of NAD-RNAs.