Source: College of Biology and Environment; Office of Research Administration

On April 7, the research team led by Professor Xia Yiji and Dr. Zhang Hailei from the Faculty of Biological and Environmental Sciences at ZWU published a paper in Nature Communications titled “High-resolution mapping reveals features of bacterial NAD-capped RNAs and stress-responsive transcription initiation.” The study established a high-precision system for identifying bacterial NAD-capped RNAs (NAD-RNA) and generated a high-resolution transcriptome-wide map of NAD-RNA in E. coli.

NAD⁺, a ubiquitous metabolic coenzyme, has recently been found to form a 5′ cap structure on RNA (NAD-cap), suggesting a role in gene expression regulation. However, existing methods suffer from two major limitations—low precision in 5′-end mapping and poor detection of short RNA species—hindering further progress in the field. To address these challenges, the team developed two complementary techniques:

1.pNADseq: This method uses an ADPRC/SPAAC reaction to efficiently and specifically label the NAD⁺ cap with biotin (with controls to eliminate background noise), followed by streptavidin enrichment and NudC-mediated decapping to expose the 5′-pA end. Adapter ligation then enables precise identification of the 5′-end adenosine of NAD-RNA, while significantly improving detection of short NAD-RNA species.

2.NADlinkSeq: Based on the same enrichment strategy, this approach converts full-length NAD-RNA into cDNA after decapping and adapter ligation, followed by Nanopore sequencing for accurate determination of full-length NAD-RNA sequences (Fig. 1).

The high-resolution map not only identified new types of NAD-RNA in E. coli—including tRNA, rRNA, and antisense RNAs—but also uncovered a conserved RDAY motif in the region adjacent to transcription start sites (positions −2 to +2). Statistically, promoters containing this motif tend to produce transcripts with higher NAD⁺ capping ratios, providing new insights into the molecular mechanisms of NAD capping (Fig. 2). Notably, under nitrogen-limited stress conditions, NAD-RNA exhibited significant TSS switching and alternative promoter usage, revealing a potential role in sensing metabolic stress and reshaping transcription initiation.

This study provides a highly accurate NAD-RNA identification strategy that can be extended to eukaryotes and presents the most comprehensive NAD-RNA map of E. coli to date, offering robust data and methodological support for understanding the biological functions of NAD⁺ capping in transcriptional regulation.

ZWU is the first affiliation of this study. The first author is Dr. Zhang Hailei, and the corresponding authors are Professor Xia Yiji, Professor Cai Zongwei (Eastern Institute of Technology, Ningbo), and Dr. Zhang Hailei. The research was supported by the ZWU Research Start-up Fund, the Research Grants Council of Hong Kong Collaborative Research Fund, and the General Research Fund, among others.