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<p>The NAD<sup>+</sup> 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 <i>E. coli</i>, 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 RNAs, 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 <i>E. coli</i> and introduces reliable methods for genome-wide profiling of NAD-RNAs across different organisms, which will facilitate the functional characterization of NAD-capping.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月7日

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  • Hailei Zhang,
  • Yinyao Qi,
  • Qiongfang Li,
  • Feng Zhang,
  • Pengxi Wang,
  • Wuzhen Liu,
  • Xiaojian Shao,
  • Bingxu Zhang,
  • Chen Zhang,
  • Yiping Wang,
  • Linfeng Huang,
  • Zongwei Cai,
  • Yiji Xia

摘要

The NAD+ 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 RNAs, 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.