High-resolution mapping reveals features of bacterial NAD-capped RNAs and stress-responsive transcription initiation

cap has been discovered in RNAs across prokaryotes and eukaryotes, suggesting a possible role of NAD capping in gene regulation. Current NAD-capped RNA (NAD-RNA) profiling methods lack precision in 5'-end mapping or bias against small NAD-RNAs. Here, we introduce precision NAD-RNA sequencing (pNAD-seq), which combines a two-step enrichment strategy with high-throughput sequencing to achieve single-nucleotide resolution of 5'-ends and unprecedented sensitivity for identifying small NAD-RNAs. We further develop NAD-linkSeq to determine full-length NAD-RNA sequences. Applying these methods to E. coli, we uncover a vast repertoire of NAD-RNAs, including tRNAs, rRNAs, intragenic transcripts, and antisense RNAs, many of which are significantly shorter than regular mRNAs, implying specialized biogenesis. High-resolution mapping reveals conserved promoter architectures driving NAD-RNA production and condition-dependent initiation dynamics: under nitrogen limitation, some NAD-RNA undergo TSS switching, alternative promoter usage, and coordinated expression shifts, correlating with metabolic stress responses. This report presents findings that offer a comprehensive view of NAD-RNAs in E. coli and introduces reliable methods for genome-wide profiling of NAD-RNAs across different organisms, which will facilitate the functional characterization of NAD-capping.