Alternative Hfq-sRNA interaction modes dictate alternative mRNA recognition

Abstract

Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.

Keywords: ChiX; Hfq; MgrR; RyhB.

Figure EV1. Effects of hfq mutations on accumulation of sRNAs

Primer extension and Northern assays shown in Fig1 together with those previously shown in Fig4 of Zhang et al (2013) (marked with asterisks). The color code for Hfq surfaces (here for the Hfq mutant allele labels) is red for the proximal surface, purple for the rim, and blue for distal surface.

Figure 1. Effect of hfq mutations on accumulation of sRNAs

Extracts were prepared from wild-type (SG30214) and isogenic hfq mutants (SG30206 to SG30237A, listed in Appendix Table S1) grown in LB medium at 37°C to early stationary phase (OD₆₀₀ ∼ 1.0). The levels of all the sRNAs were analyzed by primer extension analysis of 5 μg of total RNA. The same extracts tested in Fig4 of Zhang et al (2013) were used to analyze the sRNAs in this figure. The levels of 5S were determined by Northern analysis of the ArcZ blot in the same (Zhang et al, 2013) figure. The color code for Hfq surfaces, used throughout the paper (here for the Hfq mutant allele labels), is red for the proximal surface, purple for the rim, and blue for distal surface. We noted the presence of a second primer extension band (2–5 nt longer) for GcvB in the distal mutants. We do not know if it is due to modification or an additional transcription start site, but the band was also detected in WT cells when GcvB was expressed at high levels.

Figure 2. Effect of Hfq mutations on stability of Class I and Class II sRNAs

Wild-type and isogenic hfq mutants, deleted for the sRNA being examined and harboring a plasmid expressing the sRNA of interest under the control of P_lac, were grown at 37°C and treated as described in Materials and Methods; all transcription was stopped by treatment with rifampicin (left), or transcription of just the sRNA was stopped by washing cells to remove IPTG (right). Total RNA (3 μg for RyhB, 3.5 μg for MicF and 4 μg for all other sRNAs) extracted from each sample was subject to Northern analysis using an oligonucleotide specific to the sRNA. The levels of 5S were determined by Northern analysis of the CyaR blot. Quantitation of the Northern blots is given in FigEV2. Strains were derived from hfq⁺ (SG30214), hfqQ8A (SG30206), hfqR16A (SG30207), and hfqY25D (SG30237A) by P1 transduction to delete a given sRNA gene, as listed in Appendix Table S1.

Figure EV2. Quantitation of Northern blot analysis in Fig2

Phosphoimages of the Northern membranes were analyzed by using GE Typhoon imaging scanner and ImageJ software. Graphs were plotted with Prism software. Color code is as in Fig1. Source Data are available online for this figure.

Figure 3. A model of alternative modes of RNA binding to Hfq

The cartoon model of the Hfq hexamer depicts the three RNA-binding surfaces of Hfq: proximal face (red), rim (purple), and distal face (blue). For sRNAs and mRNAs, elements in red represent sequences (rho-independent terminator) that bind to the proximal face, elements in purple represent UA sequences that bind the rim, and elements in blue represent ARN-binding motifs that bind to the distal face. The model depicts two alternative pathways for binding and regulation by sRNA:mRNA pairs. Class I sRNAs utilize a U-rich rho-independent terminator for binding the proximal face and a UA-binding motif for interaction with the rim. mRNA targets regulated by this class of sRNAs utilize ARN-binding motifs for interacting with the distal face of Hfq. Binding of the Class I sRNA and its corresponding mRNA to Hfq lead to base pairing and regulation and degradation of the sRNA. A second class of sRNAs (Class II) utilize the U-rich rho-independent terminator for binding the proximal face of Hfq and an ARN motif for binding to the distal face. mRNA targets regulated by this class of sRNAs contain UA-binding motifs that allow for binding to the rim of Hfq. Binding of the Class II sRNA and its corresponding mRNA to Hfq lead to base pairing and regulation, but not necessarily degradation of the sRNA.

Figure 4

Role of ARN motifs in Class II sRNAs

1. Reported secondary structure of the wild-type ChiX (Rasmussen et al, 2009) and predicted secondary structure of the corresponding ARN deletion mutant (ChiXΔARN). Predicted RNA structures in all figures were determined as described in Reuter and Mathews (2010). Color codes show the ARN motifs in blue, the base-pairing region to the chiP target (Rasmussen et al, 2009) in yellow, and the U-rich region of the rho-independent terminator in red.

2. Northern blot analysis of washout experiments comparing the wild-type ChiX to the ChiXΔARN mutant. These experiments were carried out in wild-type and isogenic mutant derivatives of SG30200, each carrying ΔchiX::kan [WT hfq⁺ (DJS2784), hfqR16A (DJS2786), and hfqY25D (DJS2789)] and harboring a plasmid that expressed the wild-type ChiX (pBR-ChiX) or the ChiXΔARN mutant (pDJS2211). The washouts were carried out similarly to the experiments in Fig2, with the exception that 5 μg of total RNA was analyzed for each sample.

3. Quantitation of the Northern blot in (B). Quantification was carried out as for Fig EV2. Wild-type ChiX results are shown with a solid line and ChiXΔARN with dotted lines and WT in black, R16A in purple, and Y25D in blue.

4. β-Galactosidase activity measured in a P_BAD-chiP-lacZ ΔchiX::kan strain (DJS2991) carrying a plasmid expressing wild-type ChiX (pBR-ChiX), ChiXΔARN (pDJS2211), or a vector control (pBR-plac). Strains were grown in LB medium containing 100 μg/ml ampicillin, 10 μM IPTG, and 0.002% arabinose at 37°C to early stationary phase (OD₆₀₀ ∼ 1.0) and assayed for β-galactosidase activity. Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated.

5. Predicted secondary structure of the wild-type MgrR and the corresponding full ARN deletion mutant (MgrRΔARN) without central predicted stem-loop for MgrR. Color codes are as for Fig4A, with pairing to the eptB target (Moon & Gottesman, 2009) in yellow.

6. Northern blot analysis of washout experiments comparing wild-type MgrR to the MgrRΔARN mutant. These experiments were carried out in wild-type and isogenic mutant derivatives of SG30200, each carrying ΔmgrR::kan [WT hfq (DJS2963), hfqR16A (DJS2965), and hfqY25D (DJS2966)] and harboring a plasmid that expressed the wild-type MgrR (pBR-MgrR) or the MgrRΔARN mutant (pDJS2225). The washouts were carried out as for Fig2, with the exception that 5 μg of total RNA was analyzed for each sample.

7. Quantitation of the Northern membrane in (F) as for Fig EV2.

8. β-galactosidase activity measured in a P_BAD-eptB-lacZ ΔmgrR::kan strain (DJS3003) carrying a plasmid expressing wild-type MgrR (pBR-MgrR), MgrRΔARN (pDJS2225), or a vector control (pBR-plac). Samples were treated and analyzed as in (D), except that the LB contained 0.2% arabinose. Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated.

Source data are available online for this figure.

Figure 5

Addition of ARN motifs to a Class I sRNA

1. Reported secondary structure of the wild-type RyhB (Geissmann & Touati, 2004) and the predicted secondary structure of the chimera in which the 5′ end of ChiX was fused to truncated RyhB (ChiX-RyhB). Color codes are as for Fig4A, with region of pairing to the sodB target (Geissmann & Touati, 2004) in yellow and the ARN motifs in blue. Red nucleotides correspond to bases from RyhB, and black nucleotides correspond to bases from ChiX.

2. Northern blot analysis of washout experiments to compare the stability of the wild-type RyhB to the ChiX-RyhB chimera. These experiments were carried out in hfq⁺ ΔchiX::kan (DJS2784), hfqR16A ΔchiX::kan (DJS2786) and hfqY25D ΔchiX::kan (DJS2789), harboring a plasmid that expressed the wild-type RyhB (pBR-RyhB) or the ChiX-RyhB mutant (pDJS2219) under control of a plac promoter. The washouts were carried out as in Fig2, with the exception that 5 μg of total RNA was analyzed for each sample.

3. Quantitation of the Northern membrane in (B) as in Fig EV2.

4. β-Galactosidase activity measured in NRD537 (P_BAD-sodB-lacZ) carrying a plasmid expressing wild-type RyhB (pBR-RyhB), ChiX-RyhB (pDJS2219), or a vector control (pBR-plac). Samples were treated and analyzed as in Fig4D. Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated.

Source data are available online for this figure.

Figure 6

Activity of ChiX-RyhB chimeras in context of a Class II mRNA target

1. Secondary structures of the wild-type RyhB and the corresponding predicted secondary structures of the various ChiX/RyhB chimeras as in Fig5A.

2. β-Galactosidase activity measured in NRD537 (P_BAD-sodB-lacZ) carrying a plasmid expressing a vector control (pBR-plac), wild-type RyhB (pBR-RyhB), wild-type ChiX (pBR-ChiX), ChiX-RyhB (pDJS2219), RyhBΔUA (pDJS2227), ChiX-RyhBΔUA (pDJS2226), ChiX-RyhB-ChiX (pDJS2229), or ChiXΔARN-RyhB-ChiX (pDJS2230). Samples were treated and analyzed as in Fig4D. Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated.

3. Sequences from the 5′ UTR of P_BAD-sodB-lacZ and P_BAD-chiP+sodBbp-lacZ fusions. Color codes show the sequence for the base-pairing region to the mRNA target sodB to the sRNA RyhB highlighted in yellow and the UA element from chiP in purple. Red text represents bases from sodB, and black text represents bases from chiP.

4. β-Galactosidase activity measured in DJS2985 (P_BAD-chiP+sodBbp-lacZ) carrying the same set of plasmids as in (B). Samples were treated and analyzed as in Fig4D. Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated.

Source data are available online for this figure.

Figure 7

Sensitivity to rim and distal mutants depends on the sRNA-mRNA pair

1. Derivatives of NRD537, each with P_BAD-sodB-lacZ [WT hfq (DJS2683), hfqR16A (DJS2686), and hfqY25D (DJS2687)] and carrying a plasmid expressing wild-type RyhB (pBR-RyhB) or a vector control (pBR-plac), were grown and assayed as in Fig4D.

2. Derivatives of DJS2985, each with P_BAD-chiP+sodBbp-lacZ [WT hfq (DJS3009), hfqR16A (DJS3010), and hfqY25D (DJS3011)] and carrying a plasmid expressing wild-type RyhB (pBR-RyhB) or a vector control (pBR-plac). Samples were treated as in Fig4D.

3. Derivatives of MPK0379, each with P_BAD-csgD-lacZ ΔmcaS::kan ΔpgaA::cat [WT hfq (DJS3017), hfqR16A (DJS3018), and hfqY25D (DJS3019)] and carrying a plasmid expressing wild-type McaS (pBR-McaS), wild-type OmrA (pBR-OmrA), or a vector control (pBR-plac), were grown and assayed as in Fig4D except that the LB contained 0.02% arabinose.

Data information: Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to the vector control strain is indicated. Source data are available online for this figure.

Figure 8

Deletion of UA element in mRNA targets of Class II sRNAs leads to a loss of regulation

1. β-Galactosidase activity measured in DJS2979 (P_BAD-chiP-lacZ), DJS2982 (P_BAD-chiPΔUA-lacZ), and the ΔchiX derivatives (DJS2991 and DJS2994, respectively). Samples were treated as in Fig4D.

2. β-Galactosidase activity measured in DJS2985 (P_BAD-chiPsodBbp-lacZ) or DJS2998 (P_BAD-chiPΔUA+sodBbp-lacZ) carrying a plasmid expressing wild-type RyhB (pBR-RyhB) or a vector control (pBR-plac). Samples were treated as in Fig4D.

3. β-Galactosidase activity measured in DJS3003 (P_BAD-eptB-lacZ ΔmgrR::kan) or DJS3004 (P_BAD-eptBΔUA –lacZ ΔmgrR::kan) carrying a plasmid expressing wild-type MgrR (pBR-MgrR) or a vector control (pBR-plac). Samples were treated as in Fig4D, except that the LB contained 0.2% arabinose.

Data information: Data are average of three assays, and error bars denote the standard deviation of the mean. Percent activity compared to ΔchiX (A) or the vector control strain (B, C) is indicated. Source data are available online for this figure.

Figure 9

Addition of ARN sequences to Class II mRNA targets restores regulation by Class I but not Class II sRNAs

1. Sequences from the 5′ UTR of P_BAD-chiPΔUA+sodBbp-lacZ and P_BAD-chiPΔUA+sodBbp+rpoSARN-lacZ fusions. Color codes show the sequence for the base-pairing region to the mRNA target sodB to the sRNA RyhB highlighted in yellow, remaining sequence of the mutated UA element from chiP in purple, and ARN motifs from the 5′ UTR of rpoS in blue. Black text represents bases from chiP, and red text represents bases from sodB.

2. β-Galactosidase activity measured in DJS2998 (P_BAD-chiPΔUA+sodBbp-lacZ) or DJS3015 (P_BAD-chiPΔUA+sodBbp+rpoSARN-lacZ) carrying a plasmid expressing wild-type RyhB (pBR-RyhB), ChiX-RyhB (pDJS2219), or a vector control (pBR-plac). Samples were treated as in Fig4D, except that the LB contained 100 μM IPTG.

Source data are available online for this figure.