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.
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 Arabidopsisgenerally 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.
Detection of NAD+ in total RNA extract from Arabidopsis seedling. Total RNAs were digested with P1. The digest was separated by HPLC, and the fraction containing NAD+ was collected and analyzed by LC-MS. (A) Representative LC-MS chromatograph of the NAD+ standard, the NAD+ fraction from the P1-digested RNAs, and the control sample (the RNA sample treated with heat-inactivated P1). The experiment was repeated 3 times with similar results. (B) Product ions from NAD+ of the P1 digest were identical to those from the NAD+ standard.