CompasSeq: epitranscriptome-wide percentage assessment of metabolite-capped RNA at the transcript resolution

Nucleotide-containing metabolites, e.g., NAD, can serve as noncanonical initiating nucleotides (NCIN) during transcription, yielding NCIN-capped RNAs (NCIN-RNAs). Current profiling strategies are limited to detecting specific metabolite caps and lack an epitranscriptome-wide approach for quantifying the ratio between NCIN- and m⁷G-capped forms. Here, we develop the CompasSeq analytical platform, which integrates experimental and computational frameworks, enabling comprehensive and quantitative assessment of NCIN-RNAs at the transcript resolution. CompasSeq utilizes carefully devised enzymatic reactions to selectively capture NCIN-RNAs. By introducing proper spike-ins, CompasSeq can analyze the stoichiometry of NCIN caps. We further design an orthogonal method, the quantitative exoribonuclease reduction assay, to validate newly identified NCIN-RNAs and their capping ratios. Using CompasSeq, we quantify previously unexplored NCIN capping percentages from mouse liver and illustrate their age-associated dynamics. Moreover, we uncover a dichotomy between RNA expression and NCIN capping in genes impinging on age-related pathways. Our study presents both experimental and computational solutions for in-depth analysis of NCIN-RNAs, paving the road for functional investigations into NCIN-RNAs.