Volume 58, Issue 3, Pages 393-405 (May 2015) A 3′ External Transcribed Spacer in a tRNA Transcript Acts as a Sponge for Small RNAs to Prevent Transcriptional Noise  David Lalaouna, Marie-Claude Carrier, Szabolcs Semsey, Jean-Simon Brouard, Jing Wang, Joseph T. Wade, Eric Massé  Molecular Cell  Volume 58, Issue 3, Pages 393-405 (May 2015) DOI: 10.1016/j.molcel.2015.03.013 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of RyhB and RybB Targetomes (A) Summary of RyhB targets enriched after MS2-RyhB affinity purification (ratio MS2-RyhB/RyhB control) in an rne131 ΔryhB background. Only the reads on gene sequences are reported. (B) Northern blot analysis of grxD and erpA mRNAs. RyhB was under the control of the pBAD promoter and expression was induced by addition of 0.1% arabinose (Ara) when cultures reached an OD600nm = 0.5. As a control, the empty vector pNM12 was used. Analysis of the 16S rRNA was used as a loading control. (C) Northern blot analysis showing the effect of RyhB on GlyW, CysT, and LeuZ tRNA. RyhB expression was induced by addition of 150 μM 2,2′-dipyridyl (DIP) at an OD600 = 0.5. (D) Summary of RybB targets enriched after MS2-RybB affinity purification in an rne131 ΔrybB strain. Only the reads on gene sequences are reported. (E) Northern blot analysis showing the effect of RybB on potential or confirmed targets. RybB was under the control of the pBAD promoter, and expression was induced by addition of 0.1% arabinose when cultures reached an OD600nm = 1.0. See also Figures S1, S2, and S3. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 RyhB and RybB Pair with 3′ETSleuZ (A–C) Affinity purification with (A) MS2-RyhB, (B) MS2-RybB, and (C) MS2-3′ETSleuZ constructs. Cells were grown in LB medium to an OD600nm = 0.5, and 0.1% arabinose was added to induce expression of the control RNA (MS2-) and the MS2 tag fused to the RNA fragment of interest (MS2+) from a pBAD promoter for 10 min before cells were harvested. Northern blots and western blots (Hfq-3XFLAG; anti-FLAG antibody) were performed on samples taken before (input) and after (output) affinity purification. Co-purification of the sodB mRNA with MS2-RyhB and ompC mRNA with MS2-RybB acted as a control. Spot 42 sRNA was used as a negative control for MS2-3′ETSleuZ. (D) PbAc probing of RyhB with 3′ETSleuZ. 5′-end-labeled 3′ETSleuZ or 3′ETSmutleuZ RNAs were incubated in the absence or presence of RyhB (R) or RyhBmut (Rm) before addition of PbAc. C, non-reacted controls (lanes 1–3 and 9–11); OH, alkaline ladder (lanes 4 and 12); T1, RNase T1 ladder (lanes 5 and 13). The numbers to the left indicate sequence positions with respect to the +1 of leuZ. (E) Predicted pairing between RyhB and 3′ETSleuZ. Mutations of the 3′ETSmutleuZ and the RyhBmut are represented in red and in green, respectively. (F) In-line (MgCl2) probing of RybB with 3′ETSleuZ. 5′-end-labeled 3′ETSleuZ or 3′ETSmutleuZ RNAs were incubated in the absence or presence of RybB (R) or RybBmut (Rm) before incubating for 45 hr in the presence of MgCl2. C, non-reacted controls (lanes 1–3 and 9–11); OH, alkaline ladder (lanes 4 and 12); T1, RNase T1 ladder (lanes 5 and 13). (G) Predicted pairing between RybB and 3′ETSleuZ. Mutations of 3′ETSmutleuZ and the RybBmut are represented in red and in green, respectively. See also Figure S4. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 3′ETSleuZ Modulates RyhB Activity (A and B) β-galactosidase assays of (A) RyhB negatively regulated targets (SdhC-LacZ and FumA-LacZ translation fusion) or (B) RyhB positively regulated targets (shiA-lacZ transcriptional fusion and CirA-LacZ translation fusion) in a WT and a ΔryhB strain with an empty vector (black) or a pBAD-3′ETSleuZ (white). Cells were grown in Fe-starved minimal M63 medium containing 0.2% glycerol, in which RyhB expression is constitutive. Expression of 3′ETSleuZ was induced by addition of 0.1% arabinose when cultures reached an OD600nm = 0.1. Samples were taken at OD600nm = 0.4. (C and D) β-galactosidase assays of (C) RyhB negatively regulated targets or (D) RyhB positively regulated targets in a WT strain (black) and a 3′ETSleuZ mutant (white) grown in Fe-depleted minimal M63 medium containing 0.2% glycerol. As a control, we overproduced 3′ETSleuZ in those strains, as previously described. Data are represented as mean ± SD. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Physiological Role of 3′ETSleuZ (A) Northern blot analysis of RyhB and its targets in WT, 3′ETSmutleuZ, ΔryhB and ΔryhB 3′ETSmutleuZ backgrounds. Cells were grown in LB medium, and total RNA was extracted at an OD600nm = 0.5. The 16S rRNA was used as a loading control. (B) Succinate growth assays with WT, 3′ETSmutleuZ, ΔryhB ΔrybB, or ΔryhB ΔrybB 3′ETSmutleuZ strains in M63 minimal medium containing 0.2% succinate and 1 μM FeSO4. OD600nm was measured after 18 hr. Data are represented as mean ± SD. (C) Colicin assays in a WT, ΔryhB, 3′ETSmutleuZ, or ΔryhB 3′ETSmutleuZ strains. Cells were grown until an OD600nm = 0.1, and 1/15,000 ColIa (diluted in 50 μM HEPES buffer) was added. OD600nm was measured at various time points to monitor cell growth. Data are represented as mean ± SD. (D) Northern blot analysis of RybB and its targets in a WT, 3′ETSmutleuZ, ΔrybB and ΔrybB 3′ETSmutleuZ backgrounds. Cells were grown in LB medium, and total RNAs were extracted at an OD600nm = 1.0. See also Figure S5. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Simulation of the Effect of the sRNA Sponge on RyhB sRNA-Mediated Silencing of High- and Low-Affinity sRNA Targets (A) The red curves represent mRNA targets that pair with the sRNA more efficiently than the sRNA sponge, while blue curves represent mRNA targets that pair with the sRNA less efficiently. Solid and dashed curves show the results of simulations in the presence and absence of the sRNA sponge, respectively. The Fe-Fur concentration was changed at time zero from 1,000 nM to 5 nM and from 5 nM to 1,000 nM after 0.2 cell generation time. The deterministic differential equations that model the dynamics of the different RNA species are detailed in the Supplemental Information. (B) Northern blot analysis showing the effect of RyhB on sodB mRNA depending on its cellular concentration. RyhB expression was induced by adding 0, 50, 75, 100, and 200 μM DIP at OD600nm = 0.5 in rich medium (LB). The 16S rRNA was used as a loading control. (C) Same as in (B) but in 3′ETSmutleuZ background. Results are representative of at least three independent experiments. See also Figure S6. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Conservation of E. coli tRNA ETS and ITS Sequences in the Enterobacteriaceae Family (A) Each bar of the histogram represents the proportion of species in a given group with significant alignment with the corresponding E. coli MG1655 tRNA ETS or ITS. Colors refer to the six groups of the phylogentic tree presented in (B). tRNA coding genes were represented according to their genomic context in E. coli. The tRNA coding genes of ribosomal operons are not shown. An asterisk indicates the presence of a protein-coding gene. (B) Pre-computed phylogenetic tree of the Enterobacteriaceae. The six groups were arbitrarily defined. The number of species analyzed for each genus is indicated in parentheses. See also Figure S7. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 RybB and MicF Pair with ITSmetZ-metW and ITSmetW-metV (A) Alignment of ITSmetZ-metW and ITSmetW-metV sequences. (B–E) Affinity purification performed with (B) MS2-ITSmetZ-metW, (C) MS2-ITSmetW-metV, (D) MS2-RybB, or (E) MS2-MicF constructs. Cells were grown in LB medium to an OD600nm = 1 and 0.1% arabinose was added to induce expression of the control RNA (MS2-) and the MS2 tag fused to the RNA fragment of interest (MS2+) from a pBAD promoter for 10 min before cells were harvested. Northern blots and western blots (Hfq-3xFLAG; anti-FLAG antibody) were performed on samples taken before (input) and after (output) affinity purification. Co-purification of the ompC mRNA with MS2-RybB and ompF mRNA with MS2-MicF acted as a control. SgrS sRNA was used as a negative control for MS2-ITSmetZ-metW and MS2-ITSmetW-metV. Molecular Cell 2015 58, 393-405DOI: (10.1016/j.molcel.2015.03.013) Copyright © 2015 Elsevier Inc. Terms and Conditions