Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization and prediction of chronological age

Nicotinamide adenine dinucleotide (NAD) can be used as an initiating nucleotide in RNA transcription to produce NAD-capped RNA (NAD-RNA). RNA modification by NAD that links metabolite with expressed transcript is a poorly studied epitranscriptomic modification. Current NAD-RNA profiling methods involve multi-steps of chemo-enzymatic labeling and affinity-based enrichment, thus presenting a critical analytical challenge to remove unwanted variations, particularly batch effects. Here, we propose a computational framework, enONE, to remove unwanted variations. We demonstrate that designed spike-in RNA, together with modular normalization procedures and evaluation metrics, can mitigate technical noise, empowering quantitative and comparative assessment of NAD-RNA across different datasets. Using enONE and a human aging cohort, we reveal age-associated features of NAD-capping and further develop an accurate RNA-based aging clock that combines signatures from both transcriptome and NAD-modified epitranscriptome. enONE facilitates the discovery of NAD-RNA responsive to physiological changes, laying an important foundation for functional investigations into this modification. [Display omitted] •enONE, a computational framework of NAD-RNA, reduces technical noise using spike-in•Human peripheral blood cells contain NAD-RNA and tend to increase with age•RNA-based aging clock, integrating gene expression with NAD-RNA, can predict age•NAD-capped RNA from circulating blood can be developed as potential biomarkers Computational bioinformatics; Sequence analysis; Transcriptomics; Methodology in biological sciences