Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD ⁺ capping

Abstract

Recent findings regarding nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ noncanonical RNA capping to regulate gene expression. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to label NAD-RNAs. However, copper ions can fragment/degrade RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for labeling NAD-RNAs, followed by identification of tagged RNA by single-molecule direct RNA sequencing. We used this method to compare NAD-RNA and total transcript profiles of Escherichia coli cells in the exponential and stationary phases. We identified hundreds of NAD-RNA species in E. coli and revealed genome-wide alterations of NAD-RNA profiles in the different growth phases. Although no or few NAD-RNAs were detected from some of the most highly expressed genes, the transcripts of some genes were found to be primarily NAD-RNAs. Our study suggests that NAD-RNAs play roles in linking nutrient cues with gene regulation in E. coli.

Applications and potentials of nanopore sequencing in the (epi)genome and (epi)transcriptome era

“…149 Another way is to identify NAD-capped RNA from organelles or prokaryotic cells. 154 One problem that still needed to be resolved was to add poly(A) tails to the RNA before preparing ONT DRS libraries, as Zhang et al did. 154 The Innovation 2, 100153, November 28, 2021 www.cell.com/the-innovation…”

“…51 One way to resolve the problem is to ligate an RNA adapter to the 5 0 end of the sequenced RNAs, as was done for NAD-capped RNA with the TagSeq method. 151,154,164 The relatively higher base error rate, especially in the low-complexity genomic regions, has long prevented genetic testing and microbial detection from utilizing the full power of ONT DDS. However, the miniaturization of equipment and fast turnaround time provide hope that the two applications will become more approachable.…”

“…The enriched NAD-capped RNAs were used for ONT DRS, because the NAD-capped RNA molecules can be identified according to their adaptor sequences. 151 , 154 This strategy can greatly decrease the false-positive rate of NAD-capped RNA molecules, allowing researchers to learn the characteristics of NAD-capped RNA, e.g., whether there are differences in splicing patterns, poly(A) tail length, etc., between NAD-capped RNA and normal RNA molecules. The original protocol for labeling NAD-capped RNA was to use copper ions to catalyze click chemistry.…”

“…did. 154

Figure 6 Flowchart for ONT DRS of NAD-capped RNAs The 5′and 3′ indicate the NAD-capped RNA direction. The NAD structure was illustrated and connected to the 5′ end of the NAD-capped RNA, after ADPRC catalysis, the azide moiety was linked to NAD via replacing nicotinamide, the azide contained NAD-capped RNA then can react with SPAAC, and the synthetic RNA adapter with DBCO at its 3′ end can conjugate with azide functionalized NAD-capped RNA.

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Shaping the Bacterial Epitranscriptome—5′‐Terminal and Internal RNA Modifications

“…In addition, NAD tagSeqII—a combination of NAD tagSeq and SPAAC‐NAD‐Seq—recently identified that NAD‐capping in E. coli is dependent on its growth phases. [ 116 ]…”