NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs

Abstract

A distinctive feature of prokaryotic gene expression is the absence of 5′-capped RNA. In eukaryotes, 5′,5′-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation1. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA2. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling3,4, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5′-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH5 and against endonucleolytic cleavage by ribonuclease (RNase) E6. The nudix phosphohydrolase NudC7 decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, 13% of the abundant sRNA RNAI is NAD-capped in the presence, and 26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

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Figure 1: NAD captureSeq protocol applied to total RNA.
Figure 2: Analysis of enriched RNAs by NGS.
Figure 3: Characterization of NAD-RNAI.
Figure 4: Quantification of NAD modification of RNAI- and NAD-decapping activity of NudC in E. coli.

Accession codes

Data deposits

NGS data have been deposited at http://www.geneprof.org under accession numbers gpXP_001108, gpXP_001123 and gpXP_001153.

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Acknowledgements

We thank the CellNetworks Deep Sequencing Core Facility for Solexa sequencing, M. Brunner for access to the light cycler, M. Helm for advice on RNA mass spectrometry and B. Luisi for the RNase E expression vector, as well as A. Krause, J. Becker, A. Samanta, M. Tesch, F. Siebert, L. Obenauer and other members of the Jäschke laboratory for help and discussions. H.C. was supported by a postdoctoral fellowship from the Alexander-von-Humboldt Foundation. M.-L.W. acknowledges a PhD fellowship from the Hartmut Hoffmann-Berling International Graduate School of Molecular & Cellular Biology. A.J. is supported by the Deutsche Forschungsgemeinschaft, SFB 623, the Federal Ministry of Education and Research (BMBF), and the Helmholtz Initiative on Synthetic Biology.

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All authors designed the experiments, analysed and interpreted results and wrote the paper. H.C., M.-L.W., K.H. and G.N. performed the experiments.

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Correspondence to Andres Jäschke.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Development of the NAD capture protocol.

a, General scheme: ADPRC-catalysed transglycosylation with different alcohols, followed by ‘click’ biotinylation with biotin-PEG3-azide. b, Isolated yields and HPLC retention times. c, HPLC chromatogram of the ADPRC-catalysed reaction of NAD with 4-pentyn-1-ol. d, High-resolution ESI mass spectrum of the transglycosylation product. e, HPLC chromatogram of the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction mixture with the product from c. f, High-resolution ESI mass spectrum of the CuAAC reaction product.

Extended Data Figure 2 NAD capture protocol applied to RNA and comparison of the enrichment in NGS data sets from three different NAD captureSeq experiments.

ac, Agarose gels of the PCR amplification products of cDNA obtained from the NAD capture protocol, applied to in vitro transcribed unmodified RNA and in vitro transcribed model NAD-RNA after 5 (left), 10 (middle), and 15 (right) cycles (a); and total RNA from E. coli JM109 (b) or K-12 (c). The region (120–160 bp) around the band present in the JM109 sample (150 bp, arrow) that occurred only in the fully treated sample but not in the controls was chosen for NGS libraries of minus ADPRC control A1 and fully treated sample B1, in agreement with the expected size (<200 nucleotides) of NAD-modified RNA according to previous observations2. The intense band of 150 bp size was later attributed to the most abundant enriched RNA, RNAI, which was absent in the K-12 sample. For this reason, the K-12 sample did not contain such a prominent additional band. For comparability, the same region (120–160 bp) was excised for library preparation for K-12 control A2 and sample B2. To achieve a more comprehensive picture and study the influence of sample preparation on data, a third library was prepared from JM109 (control A3, sample B3) choosing a broader size range (50–300 bp). di, Abundance of RNAs in fully treated sample (B) versus minus ADPRC control (A) in data set 1 (JM109, 120–160 bp; d, e), data set 2 (K-12, 120–160 bp; f, g), and data set 3 (JM109, 50–300 bp; h, i) found by strand-specific NGS analysis (RPM, average of forward and reverse read counts; Supplementary Data 1–3). Triangles, enriched; circles, non-enriched.

Extended Data Figure 3 NGS analysis of specific sequences found in control A1 and sample B1 in forward and reverse reads.

Analysis of sRNAs, sRNA-like 5′-terminal fragments of mRNAs, and non-enriched tRNASer. Normalized nucleotide counts derived from NGS reads (JM109, 120–160 bp) of control A1 (red) and fully treated sample B1 (green) are plotted for the regions on the E. coli K-12 genome or the respective plasmid in which the sequences are situated. A black bar indicates the protein-coding sequence of mRNAs, while non-coding RNAs are indicated by a thin black line. Plus and minus signs designate the orientation of genes in the genome/plasmid.

Extended Data Figure 4 NGS, real-time PCR, and northern blot analysis of RNAs enriched in NAD captureSeq.

a, Enrichment of selected sequences calculated from NGS read numbers or real-time PCR data. Numbers in parentheses show enrichment of genes below abundance threshold T in the respective data set. N.D., not determined. b, Real-time PCR analysis of selected mRNAs for 5′-termini (regions found to be enriched) and regions in the middle of the mRNA. Data show that the 5′-termini are highly enriched, while middle regions are not, or are considerably less enriched. All real-time PCR experiments were performed in duplicate for the same cDNA, and no-template controls were used to assess primer dimerization. c, Northern blot analysis. The expected size range (100 to 340 nucleotides for gatY, 100 to 240 nucleotides for hdeD) is indicated and for hdeD the fragment of expected size is indicated by an arrow, d, Example of an enriched 5′-terminus, gatY. The 5′-untranslated region is indicated in grey, the coding sequence in black. Minus sign indicates that the gene is encoded on the minus-strand of the genome, thus in reverse orientation. The position of the promoter (gatYp) is indicated by an arrow. Regions amplified in real-time PCR are indicated by opposing arrows.

Extended Data Figure 5 Mass-spectrometric analysis of the ADPRC-catalysed transglycosylation reaction of NAD-RNAI from E. coli total RNA and 4-pentyn-1-ol, and analysis of the 5′-modification status of GcvB.

a, b, Disappearance of the ADP-ribose peak ([M–H] m/z = 540.05, elution at 5–6 min) (a), and appearance of a peak at m/z = 624.10 (elution time 2.0–2.5 min) upon ADPRC reaction (b). Samples were digested with nuclease P1 before mass spectrometry. c, Simulated signal pattern of the expected reaction product (isotopic distribution). df, Observed mass spectrum in the chromatography fractions eluting at 2–2.5 min of untreated RNAI (d), RNAI treated with ADPRC and 4-pentyn-1-ol and then digested by nuclease P1 (e) and 5S rRNA treated with ADPRC and 4-pentyn-1-ol and then digested by nuclease P1 (f). g, HPLC-MS extracted ion chromatograms of the peak [M]+ m/z = 664.116 of free NAD, and nuclease-P1-digested 5S rRNA and GcvB RNA. h, High-resolution mass spectra of the HPLC fractions eluting at 2.2–2.4 min.

Extended Data Figure 6 NudC processing, 5′-dependent sensitization to RNase E processing by RppH or NudC, and investigation of RNA stability in vivo depending on NudC expression.

a, b, Decapping activity on in vitro transcribed NAD-RNAI of wild-type NudC enzyme and the E178Q point mutant. Gel-electrophoretic assay, monitoring the disappearance of a site-specifically installed radiolabel (see Fig. 3c). Mean of a technical duplicate ± s.d. is shown. c, d, Successive decapping and digestion of NAD-RNAI and triphosphate-RNAI. Time course of RppH/RNase E or NudC/RNase E decapping/digests of triphosphate-RNAI (c) or NAD-RNAI (d). eh, Determination of RNAI (mean of biological duplicate ± s.d.) (e, f) and GcvB (g, h) cellular half-life depending on NudC expression by rifampicin stop assay. Detection by northern blotting, tRNASer as loading control. Source data

Extended Data Figure 7 Determination of the 5′-modification status of cellular RNAs by a modified PABLO assay5,40.

a, Schematic overview of NAD quantification by dephosphorylation, NudC cleavage and splinted ligation. Total RNA is isolated from different E. coli strains: wild type + RNAI (1); ΔnudC + RNAI (2); ΔnudC + NudC + RNAI (3); or ΔnudC + NudC(E178Q) + RNAI (4) (for strain details, see i). To remove all 5′-monophosphate and 5′-triphosphate termini, total RNA samples are subjected to alkaline phosphatase treatment. Then, to remove 5′-NAD and produce 5′-monophosphate termini, the dephosphorylated samples are treated with NudC (or NudC(E178Q) as a negative control). Finally, the resulting 5′-monophosphate RNA is ligated specifically to an adaptor by splinted ligation. Ligated fractions are determined by northern blotting. b, c, Validation of time-dependent in vitro NudC processing of cellular sRNAs (RNAI, 6S RNA) in total RNA from E. coli wild type + RNAI (1), using the assay described in a. Aliquots were taken after different times of NudC treatment and subjected to ligation as shown. tRNASer is blotted as loading control. d, e, Quantification of the efficiency of NudC treatment and splinted ligation (processing efficiency) for NAD-full-length RNAI (NAD-FL-RNAI) and NAD-6S RNA. To calculate the percentage of NAD modification (shown in Fig. 4), it is necessary to normalize to the cumulative processing efficiency of NudC treatment and splinted ligation of NAD-RNA, which is determined experimentally for each total RNA preparation. To this end, in vitro transcribed radiolabelled NAD32P-FL-RNAI (d) or NAD32P-6S RNA (e) is spiked into total RNAs isolated from the four E. coli strains mentioned above. The mixture is then subjected to the treatment described in a. Processing efficiencies of radiolabelled RNAs are quantified ratiometrically after gel electrophoresis and electroblotting. f, g, Quantification of the percentage of RNAI−5. Cellular RNAI consists of a mixture of full-length and processed species, among them RNAI−5 (ref. 42), which arises from RNase E cleavage, thus contains a 5′-monophosphate, and lacks only five 5′-terminal nucleotides. Both species exhibit very similar electrophoretic mobility. Here, we were interested only in the percentage of NAD-modified FL-RNAI. Thus, we quantified the percentage of RNAI−5 by PABLO analysis (splinted ligation of non-treated total RNA with RNAI−5-specific adaptor). Carrying out the respective control (in vitro transcribed monophosphate32P-RNAI−5, spiked into total RNA samples) (f), we determined the percentage of ligation product, and normalized to ligation efficiencies to obtain the percentage of RNAI−5 (g). With the help of the values determined in dg, we then determined the percentage of FL-RNAI and the percentage of NAD-FL-RNAI (shown in Fig. 4a), using the formulas shown in h. Values shown in this figure represent replicate 1. (Expected) ligation products are marked with arrows (bg). i, Strains used in this study.

Extended Data Table 1 Listing of all enriched high-abundance sequences from three different NAD captureSeq experiments

Supplementary information

Supplementary Information

This file contains Supplementary Table 1. (PDF 178 kb)

Supplementary Data 1

This file contains strand-specific and non-strand-specific sequence count analysis for data set 1. (XLSX 1937 kb)

Supplementary Data 2

This file contains strand-specific and non-strand-specific sequence count analysis for data set 2. (XLSX 3915 kb)

Supplementary Data 3

This file contains strand-specific and non-strand-specific sequence count analysis for data set 3. (XLSX 1782 kb)

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Cahová, H., Winz, M., Höfer, K. et al. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature 519, 374–377 (2015). https://doi.org/10.1038/nature14020

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