Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle

Nicotinamide adenine dinucleotide (NAD), a nucleotide-containing metabolite, can be incorporated into the RNA 5'-terminus to result in NAD-capped RNA (NAD-RNA). Since NAD has been heightened as one of the most essential metabolites in cells, its attachment to RNA might indicate a yet-to-be discovered mechanism at the epitranscriptomic level. Here, we design a highly-sensitive method, DO-seq, to capture NAD-RNAs. Using Drosophila, we identify thousands of previously unexplored NAD-RNAs and their dynamics in the fly life cycle, from embryo to adult. We show the evidence that chromosomal clustering might be the structural basis by which co-expression can couple with NAD capping on physically and functionally-linked genes. Furthermore, we note that NAD capping of cuticle genes seems to inversely correlate with gene expression. Combined, we propose NAD-RNA epitranscriptome as a hidden layer of regulation that profoundly impacts biological processes. DO-seq empowers the identification of NAD-capped RNAs, facilitating functional investigation into this modification. Overall design: DO-seq of total RNAs (2-month-old mouse livers) with spike-in RNA that had different ratios of NAD-RNA (0, 1, 5%); DO-seq with or without yDcpS treatment of mouse livers (18-month-old); NAD-RNA profiles and transcriptome profiles of Drosophila life cycle (embryo (14h-16h AEL), larva (4d AEL), pupa (7d AEL) and adult fly (3d)) were generated by deep sequencing, using Illumina Novaseq.To profile NAD-RNAs from Drosophila life cycle, total RNAs from those samples were mixed with spike-in RNA from mouse livers, and two synthetic spike-in, of which one with 5% NAD-capped forms and another with 100% m7G-capped forms. The mixtures were then subjected to DO-seq.