Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization

ABSTRACT The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA transcription to result in NAD-capped RNA (NAD-RNA). NAD-RNA that intimately connects metabolite with gene expression can be developed as novel biomarkers for aging and disease. Epitranscriptome-wide profiling of NAD-RNAs involves chemo-enzymatic labeling and affinity-based enrichment; yet currently available computational analysis cannot adequately remove variations associated with capture procedures. Here, we propose a spike-in-based normalization and data-driven evaluation framework, enONE, for the omic-level analysis of NAD-capped RNAs. We demonstrate that carefully designed spike-in RNAs, together with modular normalization procedures and evaluation metrics, can lead to the optimal normalization that maximally removes unwanted variations, empowering quantitative and comparative assessment of NAD-RNAs from different datasets. Using enONE and a human aging cohort, we reveal critical features of NAD-capped RNAs that occur with normal age. enONE facilitates the discovery of NAD-capped RNAs that are responsive to physiological changes, laying a critical foundation for functional investigations into this modification.