NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes

Two methods NADcapPro and circNC are presented for precise identification of NAD-caps and non-canonical caps in eukaryotic RNAs. Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m(7)G-capped RNAs can be different, and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mitochondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.

Automated summary

Two methods, NADcapPro and circNC, are introduced to precisely identify NAD-capped RNAs and non-canonical caps in eukaryotes. These methods overcome the limitations of previous techniques and reveal unexpected features of NAD-capped RNAs in budding yeast. The study shows that NAD-RNAs can be full-length, polyadenylated transcripts, and that their transcription start sites can be different from canonical m(7)G-capped RNAs. Additionally, NAD caps can be added after transcription initiation, and NAD-RNAs exhibit a preference for translation in mitochondria.