1. Volume 24, Issue 7, Pages 1890-1901.e8 (August 2018)
Identification, Biosynthesis, and Decapping of NAD-Capped RNAs in B. subtilis 
Jens Frindert, Yaqing Zhang, Gabriele Nübel, Masroor Kahloon, Leonie Kolmar, Agnes Hotz-Wagenblatt, Jürgen Burhenne, Walter E. Haefeli, Andres Jäschke 
Cell Reports 
Volume 24, Issue 7, Pages e8 (August 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 1890-1901.e8DOI: (10.1016/j.celrep.2018.07.047)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
NAD captureSeq Applied to Total RNA, Analysis of the NGS Data, and qPCR Analysis of Enriched and Non-enriched RNAs
(A) Abundance of RNAs in the ADPRC-treated sample (S) versus the minus ADPRC control (NC). Each RNA is depicted as a dot in the plot. Green dots have a false discovery rate (FDR) < 0.1; gray dots have an FDR ≥ 0.1.
(B) Classification of RNAs found by NAD captureSeq. See Figure S1F for the actual numbers.
(C) Comparison of the 252 RNAs enriched by NAD captureSeq (normalized base mean > 10, log2 fold change > 2, FDR < 0.1, and pval < 0.05) and the 252 highest-expressed RNAs in B. subtilis under the same growth conditions (data from Nicolas et al., 2012).
(D) Genome Browser tracks (Nicol et al., 2009) of the enriched veg mRNA and the non-enriched hag mRNA. Normalization is in counts per million mapped reads.
(E) Design of the qPCR experiment using the cDNA library. NGS adapters are marked in magenta and cyan. Primers are depicted as arrows. 5′ (m), 5′ end (middle part) of the respective RNA.
(F) Table of RNAs enriched and non-enriched in the NAD captureSeq NGS data and on the cDNA level. Enrichment was calculated using the ΔΔCT method (Livak and Schmittgen, 2001).
See also Figures S1 and S2.
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4. Figure 2
Incorporation of NAD into veg mRNA by In Vitro Non-canonical Transcription Initiation of RNA Polymerase
(A) Incorporation of 32P-NAD into veg in the absence or presence of sigma factor A or B. M, RNA size standard.
(B) Promoter region of the veg mRNA and its −1 mutants. The starting nucleotide is marked with an arrow.
(C) Effect of the −1 position of the promoter on transcription efficiency. Reactions were performed in the presence of α-32P-ATP with or without sigma factor A. Reaction products were separated by PAGE. Quantification is mean ± SD, n = 3 (technical replicates).
(D) Effect of the −1 position of the promoter on NAD incorporation into RNA. Reactions were performed as for (C) but in the presence of NAD (the ATP:NAD ratio was 1:1). Reaction products were separated on APBgels. Quantification as in (C).
See also Figures S4 and S5.
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5. Figure 3
Influence of the −1 Promoter Position (T-to-C Transition) and of the Rifampicin Binding Pocket on NAD-RNA Formation in B. subtilis
(A) Total RNA was isolated and separated on an APBgel, and the veg mRNA was detected by northern blot analysis (upper part). Wild-type (WT), B. subtilis 1A1; Δveg+veg, B. subtilis Δveg + pHT01-Pveg-veg; Δveg+veg-1C, B. subtilis Δveg + pHT01-Pveg-veg-1C; Δveg, B. subtilis Δveg; NAD-veg, in vitro transcribed NAD-veg mRNA; 5S rRNA, loading control. Biological replicates of B. subtilis Δveg+veg and B. subtilis Δveg+veg-1C (lower part).
(B) Quantification of NAD-veg mRNA from (A) (lower part); mean ± SD, n = 3 (biological replicates).
(C) Effect of mutations rpoB-Q469I and rpoB-H482Y of the rifampicin-resistant RNA polymerases on NAD incorporation into RNA. Reactions were performed in the presence of α-32P-ATP, sigma factor A, and NAD (ATP:NAD ratio of 1:1). Reaction products were separated on an APBgel.
(D) Quantification of the NAD- and ppp-veg-mRNA of the experiment described in (C). Statistics are as in (B).
(E) Quantification of the NAD- and ppp-veg-mRNA of the experiments described in (C) with a ATP:NAD ratio of 1:4. In addition, the reaction time was decreased from 30 to 5 min. Statistics are as in (B).
See also Figure S5.
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6. Figure 4 BsRppH Decaps NAD-RNA In Vitro
(A) 32P-NAD-veg mRNA was incubated with BsRppH or NudC in Mn2+ or Mg2+ buffer. Half of the samples were subsequently treated with nuclease P1 (Nuc. P1). The reaction products were resolved by TLC.
(B) NAD-veg mRNA was incubated in Mn2+ buffer with BsRppH, BsRppH-E72Q, EcRppH, or no enzyme. Reaction products were separated on APBgels.
(C) Quantification of the NAD-veg mRNA from (B); mean ± SD, n = 3 (technical replicates).
(D) Nudix motif of BsRppH is responsible for NAD-RNA decapping. Assay as in (A).
(E) Proposed decapping mechanism of NAD-RNA by BsRppH. The red P highlights the position of the 32P.
See also Figures S6 and S7.
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7. Figure 5
Sequence and Structure Requirements for NAD-RNA Decapping by BsRppH
(A) 5′-NAD-NU 5S rRNA. N, any of the four nucleotides.
(B) 5′-NAD-5S rRNAs with different nucleotides at the +2 position were incubated with BsRppH (RNA:enzyme ratio of 1:2) in Mn2+ buffer. Reaction products were separated on APBgels.
(C) Quantification of the NAD-5S rRNAs from (B); mean ± SD, n = 3 (technical replicates).
(D) 5′-double-stranded 5′-NAD-5S rRNA (upper part) and 5′-single-stranded 5′-NAD-GU 5S rRNA were treated as in (B).
(E) Quantification of the NAD-RNA from (D). Statistics are as in (C).
See also Figures S6 and S7.
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8. Figure 6
BsRppH Prefers NAD-veg RNA over Free NAD+ In Vitro, and Its Absence Affects the Expression of Hundreds of Genes In Vivo
(A) NAD-veg mRNA was incubated with BsRppH in the absence or presence of 1,000-fold (0.1 mM) or 10,000-fold (1 mM) excess of NAD+ in Mn2+ buffer. Reaction products were separated on APBgels.
(B) Quantification of the NAD-veg mRNA from (A).
(C) Volcano plot of transcripts comparing wild-type (WT) and BsRppH-depleted (ΔmutTA) samples. The log2 fold change (ΔmutTA/wt) is plotted versus the −log2 FDR (false discovery rate). Every transcript is depicted as a dot in the plot (base mean > 100, log2 fold change > 2 (red) (<−2 (blue)), −log2 FDR > 100).
(D) Functional clustering of gene products of enriched RNAs (red dots in (C)) using DAVID (Huang et al., 2009) and FGNet (Aibar et al., 2015). Colored lines mark the clusters. Candidates belong to one (colored circle) or more (white circles) clusters. Gene ontology (GO) and InterPro annotation was used for the creation of the clusters. Terms marked with an asterisk (∗) are in several clusters.
(E) Comparison of the SigB regulon RNAs with RNAs enriched in ΔmutTA cells.
See also Figures S6 and S7.
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9. Figure 7
The NAD-Cap Stabilizes veg mRNA from 5′-to-3′-Exonucleolytic Degradation of RNase J1
(A) ppp-veg and NAD-veg mRNA (both body labeled) and p-veg mRNA (5′ labeled) were incubated with RNase J1. Reaction products were separated by PAGE. ∗, RNase J1-H76A; M, RNA size standard.
(B) Quantification of the RNA from (A); mean ± SD, n = 3 (technical replicates).
(C) Proposed effect of the 5′ status of RNA on the activity of RNase J1.
See also Figure S7.
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