A small RNA activates CFA synthase by isoform-specific mRNA stabilization
2013; Springer Nature; Volume: 32; Issue: 22 Linguagem: Inglês
10.1038/emboj.2013.222
ISSN1460-2075
AutoresKathrin S. Fröhlich, Kai Papenfort, Ágnes Fekete, Jörg Vogel,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoArticle18 October 2013free access Source Data A small RNA activates CFA synthase by isoform-specific mRNA stabilization Kathrin Sophie Fröhlich Kathrin Sophie Fröhlich RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Kai Papenfort Kai Papenfort RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Agnes Fekete Agnes Fekete Pharmaceutical Biology, Julius-von-Sachs-Institute of Biosciences, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Jörg Vogel Corresponding Author Jörg Vogel RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Kathrin Sophie Fröhlich Kathrin Sophie Fröhlich RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Kai Papenfort Kai Papenfort RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Agnes Fekete Agnes Fekete Pharmaceutical Biology, Julius-von-Sachs-Institute of Biosciences, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Jörg Vogel Corresponding Author Jörg Vogel RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Author Information Kathrin Sophie Fröhlich1, Kai Papenfort1, Agnes Fekete2 and Jörg Vogel 1 1RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany 2Pharmaceutical Biology, Julius-von-Sachs-Institute of Biosciences, Biocenter, University of Würzburg, Würzburg, Germany *Corresponding author. Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany. Tel.:+49 931 3182575; Fax:+49 931 3182578; E-mail: [email protected] The EMBO Journal (2013)32:2963-2979https://doi.org/10.1038/emboj.2013.222 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. Introduction The intense study of eukaryotic microRNAs and bacterial small regulatory RNAs (sRNAs) over the last decade has provided tremendous insight into how small RNAs use base-pairing mechanisms to control large post-transcriptional regulons. These studies focussed predominantly on the repression of mRNAs targets, which is the primary mode of action of both the eukaryotic microRNAs and the bacterial sRNAs (Lioliou et al, 2010; Storz et al, 2011; Pasquinelli, 2012). The bacterial base-pairing sRNAs include the prominent class of Hfq-associated sRNAs, which are regarded as the largest group of post-transcriptional regulators in Gram-negative bacteria (Vogel and Luisi, 2011; De Lay et al, 2013). Model bacteria such as Escherichia coli and Salmonella enterica (hereafter referred to as Salmonella) may express >100 sRNAs (Chao et al, 2012; Zhang et al, 2013), most of which act on multiple targets in a large set of cellular pathways. Hfq, the RNA-binding protein from which this class of sRNAs derives its name, has multiple functions in this regulation: it stabilizes many sRNAs prior to target recognition, generally facilitates the short seed interactions between sRNAs and mRNAs, and recruits auxiliary factors such as the major mRNA decay enzyme RNase E (Morita and Aiba, 2011; Vogel and Luisi, 2011). Regarding the mechanisms of target repression by Hfq and sRNAs, current evidence supports two general scenarios, namely the inhibition of translation and direct mRNA destabilization. In the former case, an sRNA binds near its target's ribosome binding site (RBS) to sterically interfere with 30S ribosome association. This primary event of translational repression may subsequently be rendered irreversible through mRNA decay either by concomitant recruitment of RNase E as part of a tripartite sRNA:Hfq:RNase E complex (Morita et al, 2005; Prevost et al, 2011), or simply by the increased vulnerability of the untranslated mRNA to the RNase E-containing degradosome (Belasco, 2010). As a variation on the theme, translation may be repressed by sRNA-guided loading of Hfq near the RBS rather than by the sRNA-mRNA duplex itself (Desnoyers and Masse, 2012). In the other general scenario, translational control is bypassed and mRNA destruction is the primary event in target repression. In this case, RNase E is recruited to either the sRNA-mRNA duplex itself, or a nearby site that becomes accessible due to a structural rearrangement in the target mRNA (Afonyushkin et al, 2005; Desnoyers et al, 2009; Pfeiffer et al, 2009; Bandyra et al, 2012; Mackie, 2013a). In contrast to eukaryotic microRNAs that almost exclusively cause mRNA repression, bacterial sRNAs are also known to activate mRNAs. A recurring theme among Hfq-associated sRNAs has been an anti-antisense mechanism whereby the sRNA acts as a competitive binder to prevent the formation of an intrinsic inhibitory structure around the RBS of the target mRNA. As a result, protein synthesis is upregulated, and the increased translation usually indirectly stabilizes the target mRNA (Fröhlich and Vogel, 2009; Soper et al, 2010). Other positive modes of regulation by Hfq and sRNAs have been reported too. For example, the cis-encoded antisense GadY RNA of E. coli promotes RNase III-mediated cleavage of the dicistronic gadXW mRNA, resulting in a more stable monocistronic gadX transcript and higher synthesis of GadX protein (Opdyke et al, 2004, 2011). In E. coli and Salmonella, there is also positive regulation in trans by the chitobiose operon mRNA that traps the ChiX/MicM sRNA and prevents the latter from repressing an unrelated chitoporin mRNA (Figueroa-Bossi et al, 2009; Overgaard et al, 2009). A different decoy mechanism for indirect activation was recently shown to control biofilm formation in E. coli. Here, the sRNA McaS sequesters the RNA-binding protein CsrA to alleviate CrsA-mediated translational repression of the pgaA mRNA (Jorgensen et al, 2013). Notwithstanding these diverse modes of positive regulation, it is surprising that a pathway whereby sRNAs activate targets by direct interference with the RNase E-mediated decay has not been reported until recently. RNase E degrades many cellular mRNAs by recognizing the 5′ end, that is, the region of the mRNA where sRNAs commonly bind, followed by endonucleolytic cleavage and subsequent 3′ to 5′ exonucleolytic degradation by other enzymes (Belasco, 2010; Mackie, 2013b). Moreover, mRNA duplexes—both intrinsic and resulting from cis-antisense transcription in the 5′ UTR—can protect mRNAs from RNase E (Bouvet and Belasco, 1992; Stazic et al, 2011). Similarly, there are several reports of positive regulation in which 30S ribosomes or CsrA occlude RNase E recognition sites in the 5′ UTR of an mRNA (Braun et al, 1998; Lodato et al, 2012; Yakhnin et al, 2013). Collectively, this led us to hypothesize the existence of a translation-independent activation mechanism whereby Hfq and sRNAs act to activate targets directly by site-specific interference with RNase E-mediated mRNA decay. In support of this hypothesis, we have recently reported that the Hfq-associated SgrS sRNA selectively activates the synthesis of the phosphatase YigL by the selective capture and stabilization of an RNase E decay intermediate of the pldB-yigL operon mRNA (Papenfort et al, 2013). Here, we present evidence for a direct activation mechanism that operates in the 5′ region of a full-length monocistronic mRNA. We report that the conserved sRNA RydC activates the synthesis of cyclopropane fatty acid (CFA) synthase in Salmonella. RydC was originally discovered by its co-immunoprecipitation (coIP) with Hfq in E. coli (Zhang et al, 2003). In vitro structure probing predicted an unusual pseudoknot structure of this sRNA, and an initial functional analysis suggested that RydC may be involved in the regulation of the yejABEF mRNA which encodes a putative ABC transport system (Antal et al, 2005). However, direct targets of RydC have been unknown, as has a conserved function of this sRNA. Using a combination of biochemical and genetic approaches, we demonstrate that RydC pairs with the 5′ UTR of a longer isoform of cfa mRNA. Formation of this RNA interaction, which occurs far upstream of the cfa start codon, is independent of the actual seed sequence of RydC, alters RNase E-dependent decay and stabilizes the target, even in the absence of mRNA translation. There is also evidence to suggest that independent of the base pairing, the sRNA-guided recruitment of the Hfq protein as an effector contributes to stabilization of the cfa mRNA. Physiologically, RydC may be the first regulatory sRNA known to influence bacterial membrane stability. Results Expression and molecular architecture of RydC RydC is an ∼65-nt sRNA originally discovered in E. coli (Zhang et al, 2003; Antal et al, 2005) whose sequence and 3′ flanking gene (cybB) are conserved in many other enterobacterial species (Figure 1A; Supplementary Figure S1). Northern blot analysis of RydC in Salmonella revealed its expression throughout growth in L-broth, and as single RNA species with the length predicted for the primary RydC transcript (Figure 1B). The expression profile of RydC matches its previously reported association pattern with Hfq (Chao et al, 2012). Using in vitro synthesized RNA as a concentration standard, we estimated that under the conditions tested, there were ∼4–16 copies of RydC present per cell (Figure 1B). Figure 1.Conservation, structure and expression of RydC. (A) Alignment and pseudoknot structure of RydC RNA. STM: Salmonella Typhimurium; SBG: Salmonella bongori; CKO: Citrobacter koseri; ECO: Escherichia coli; SFL: Shigella flexneri; EFE: Escherichia fergusoni; EAE: Enterobacter aerogenes; KPN: Klebsiella pneumoniae. Mutations K1, K2 and K1/2 introduced to alter pseudoknot formation are indicated. (B) RydC levels in total RNA samples (corresponding to 1 OD600) of wild-type Salmonella at indicated time points of growth were compared on northern blots to signals of in vitro transcribed RydC to estimate the in vivo copy number. 5S RNA served as a loading control. (C) Expression levels of RydC, RydC-K1, RydC-K2 and RydC-K1/2 (as described in (A); expressed from the constitutive PL promoter) were determined in ΔrydC Salmonella by northern blot analysis of total RNA samples (OD600 of 1). (D) Stabilities of RydC, RydC-K1, RydC-K2 and RydC-K1/2 were determined in ΔrydC Salmonella by northern blot analysis of total RNA samples withdrawn prior to and at indicated time points after inhibition of transcription by rifampicin at an OD600 of 1. See Supplementary Figure S4 for quantification.Source data for this figure is available on the online supplementary information page. Source Data for Figure 1 [embj2013222-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint RydC is a representative example of a Hfq-associated sRNA with a potential pseudoknot fold (Antal et al, 2005), which is a structural motif more commonly found in larger transcripts or tRNAs (Brierley et al, 2007). While this pseudoknot was inferred from in vitro structure probing and sequence comparison (Antal et al, 2005), its impact on RydC expression or function in vivo remained unknown. Our alignment of currently available rydC sequences suggests an intriguing architecture whereby several compensatory mutations have structurally maintained a central core based on two pseudoknot helices, which are preceded by 10 ultra-conserved single-stranded nucleotides at the 5′ end of the sRNA (Figure 1A; Supplementary Figure S2). To address the relevance of the pseudoknot fold in vivo, we disrupted helix 1 by changing the two conserved guanosines at positions 37 and 39 to cytosines (mutant RydC-K1; Figure 1A; Supplementary Figure S3). We observed an ∼5-fold decrease in RNA steady-state levels compared to wild-type RydC (Figure 1C), which was accompanied by a reduction in the half-life of the RNA from >32 min to 3-fold change (P-value<0.15) are marked in red. (B) RydC and cfa mRNA levels were determined on northern blots of total RNA extracted from rydC mutant cells carrying plasmids pBAD or pBAD-RydC at indicated time points prior to and after addition of arabinose (Ara). The oligo directed against the 5′ UTR of cfa specifically recognizes cfa1 mRNA. (C) Expression of Cfa-3xFLAG in wild-type and ΔrydC mutant Salmonella either carrying a control construct or a plasmid for the constitutive overexpression of RydC from the PL promoter was monitored over growth on western blots. (D) Total ion chromatograms of Salmonella wild-type cells carrying a control plasmid or a ΔrydC mutant transformed with the RydC overexpression plasmid pPLRydC. Cells were grown in M9 minimal medium to exponential phase (OD600 of 0.5), and after alkaline hydrolysis, total fatty acids were analysed by LC/MS. Peaks assigned to C16UFA, C17CFA, C18UFA and C19CFA are indicated. (E) Relative quantification of C16UFA, C17CFA, C18UFA and C19CFA in Salmonella Δcfa or ΔrydC carrying either a control plasmid or pPLRydC. All measurements were normalized to wild type; error bars represent the standard deviation calculated from three independent biological replicates; nd: not detected.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013222-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint We focussed on cfa as the target showing strongest regulation. A time-course experiment in which changes in cfa mRNA levels were monitored by northern blot analysis revealed the activation to be rapid, leading to ∼2.2-fold and ∼7.3-fold higher cfa mRNA levels at 2 and 15 min post pBAD-RydC induction, respectively (Figure 2B, lanes 4–8). This activation was specific to RydC, that is, not seen with the empty pBAD control vector (Figure 2B, lanes 1–3). To monitor Cfa protein levels by quantitative western blot, we tagged the cfa gene in the Salmonella chromosome with a C-terminal 3 × FLAG epitope. While the levels of Cfa protein did not significantly differ between the wild-type and ΔrydC strains at various stages of growth examined (likely due to low basal expression of RydC; Figure 2C, lanes 1–4 versus 5–8), overexpression of RydC increased the abundance of this protein up to ∼11-fold (lanes 1–4 versus 9–12). This observed increase in Cfa protein was corroborated by measuring the effect of RydC expression on cellular lipid composition. The CFA synthase converts the cis double bond of pre-existing unsaturated fatty acids (UFAs) of membrane phospholipids into a more stable methylene bridge (Grogan, 1997). This modification increases the stability of the bacterial membrane and can be detected by liquid chromatography/mass spectrometry (LC/MS) analysis (Nagy et al, 2004). In comparing the total fatty acid content by LC/MS, using a Salmonella cfa mutant as a negative control, we detected an ∼4-fold increase in CFA (C17CFA; C19CFA) levels in the RydC overproducing strain, as compared to the wild-type (Figure 2D and E). Thus, RydC can affect bacterial membrane composition through activation of Cfa protein. RydC selectively activates the longer of two cfa mRNA isoforms Previous studies have identified two conserved transcription start sites (TSSs) for cfa in E. coli, Salmonella and other related γ-proteobacteria (Wang and Cronan, 1994; Kim et al, 2005), and both TSSs were confirmed in a recent dRNA-seq analysis of the Salmonella strain SL1344 used in this study (Kröger et al, 2012). Transcription from the distal TSS associated with a σ70-dependent promoter yields the cfa1 mRNA with an unusually long (210 nt) 5′ UTR. A shorter mRNA, cfa2, originates from the proximal TSS, 33 bp upstream of the start codon (Figure 3A; Supplementary Figure S6). Transcription of cfa2 requires the major stress σ-factor σS, encoded by rpoS, and is considered to account for the increased Cfa levels in stationary phase bacteria (Cronan, 2002). Figure 3.RydC acts on one of the two isoforms of cfa mRNA. (A) Sequence of the Salmonella cfa upstream region. Transcription initiates from two start sites indicated by arrows at −210 bp (σ70-dependent; cfa1 mRNA) or −33 bp (σS-dependent; cfa2 mRNA) relative to the translational start site, respectively. Promoter elements are highlighted in grey, and the start codon is boxed. (B) RydC specifically acts on the longer cfa mRNA isoform. Salmonella cfa::3xFLAG ΔrydC cells or an isogenic ΔrpoS mutant were transformed with the pBAD control plasmid (−) or the pBAD-RydC overexpression plasmid (+); Salmonella Δcfa ΔrydC served as a negative control. All strains were grown to an OD600 of 2, and total RNA samples withdrawn prior to and 15′ after arabinose addition were used as templates for primer extension. Transcripts originating from either TSS1 or TSS2 were identified using gene-specific sequencing ladders. (C) Schematic representation of the cfa gene including the upstream promoter region. Translational cfa::gfp fusions (under control of the constitutive PLTetO-1 promoter) were constructed comprising the 5′ upstream region from the distal (cfa1::gfp) or the proximal start site (cfa2::gfp) plus the first 45 nucleotides of the cfa CDS. (D) Regulation of reporter fusions was monitored by western blot analysis. At an OD600 of 1, total protein samples were prepared from Salmonella ΔrydC ΔrpoS mutants carrying plasmids to express cfa2::gfp or cfa1::gfp in combination with a control plasmid (−) or pPLRydC (+). GroEL served as a loading control. Expression of RydC was validated on a northern blot.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013222-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint To understand which of the two isoforms of the cfa mRNA is controlled by RydC, we examined their abundance using primer extension experiments, prior to and 15 min after pBAD-RydC induction. Strikingly, RydC significantly activated the longer cfa1 mRNA but had no effect on the shorter cfa2 transcript (Figure 3B, lanes 1–4). As expected, activation of the σ70-dependent cfa1 mRNA was unaffected by a ΔrpoS mutation; by contrast, the σS-dependent cfa2 transcript was no longer detected in the ΔrpoS strain, with or without RydC (lanes 5–8). Importantly, the activation of cfa1 expression in wild-type cells reflects the effect of RydC on the total cfa mRNA pool, that is, the sum of products from both TSSs. Using quantitative real-time PCR (qRT–PCR) and primers binding in the codon sequence (which is common to both the cfa1 and cfa2 transcript) total cfa mRNA levels were observed to increase ∼3-fold upon RydC expression for 15 min (unpublished results). The rapid activation of the cfa mRNA by RydC (Figure 2B) suggested control at the mRNA level. To address this, we cloned the 5′ UTRs of cfa1 or cfa2, along with the first 15 codons, into a GFP reporter plasmid (Urban and Vogel, 2007) and tested reporter activation by RydC. Note that these GFP reporters are transcribed from a constitutive (PLtetO) promoter, so changes in reporter activity indicate post-transcriptional regulation. Again, RydC overexpression did not affect the cfa2::gfp reporter but upregulated the cfa1::gfp reporter 7-fold (Figure 3D); similarly, the levels of only the cfa1::gfp and not the cfa2::gfp mRNA increased in the presence of RydC (Supplementary Figure S7). In further support of post-transcriptional regulation, an intact hfq gene was essential for the observed activation (Supplementary Figure S8). Interestingly, basal reporter activity of the cfa1::gfp construct was ∼10-fold lower than that of cfa2::gfp (Figure 3D, lane 1 versus 3). Thus, the shorter cfa2 mRNA yields higher levels of Cfa protein, whereas the long 5′ UTR of the cfa1 mRNA limits Cfa synthesis. However, using a cfa1-specific mechanism of post-transcriptional activation, RydC can achieve the same levels of Cfa synthesis as stress-induced transcription from the cfa2-associated σS promoter. RydC activates Cfa expression by seed pairing Given that mRNA regulation by Hfq-associated sRNAs typically involves base-pairing interactions (Vogel and Luisi, 2011), we experimentally and biocomputationally searched for a potential RydC binding site in the cfa1 mRNA. First, we subjected a radiolabelled, ∼300 nt RNA fragment (from the distal cfa TSS to residue 70 of the CDS) to structure probing with lead(II) acetate or RNase T1. Addition of RydC did not induce significant changes in the cleavage patterns in the cfa mRNA fragment (Figure 4A, lanes 4 versus 6 and 8 versus 10), likely because it requires Hfq for annealing to the mRNA. Indeed, concomitant addition of RydC and Hfq—but not Hfq alone—protected region −99 to −109 nt relative to the cfa start codon (lanes 7 and 11). This putative RydC site lies ∼60 nt upstream of where the cfa2 mRNA begins and is consistent with the observed selective regulation of cfa1 mRNA. Figure 4.RydC employs its conserved, single-stranded 5′ end to base pair with cfa1 mRNA. (A) In vitro structure probing using 5′ end-labelled cfa mRNA (TSS1 to nt 70 of the CDS; 20 nM) with lead(II) acetate (lanes 1–4) and RNase T1 (lanes 5–8) in the presence and absence of Hfq (20 nM) and RydC (200 nM). RNase T1 and alkaline ladders of cfa mRNA were used to map cleaved fragments. Positions of G-residues are indicated relative to the translational start site. The RydC binding site and the Shine-Dalgarno (SD) region are marked. (B) Predicted duplex forming between RydC (nts 2–11) and cfa mRNA (nts −109 to −99 relative to the translational start site). Positions of single-nucleotide exchanges generating the compensatory mutants RydC* and cfa* mRNA are indicated. (C) Schematic representation of wild-type MicA and RydC as well as the derivative constructs. The first 13 nts of RydC were fused to the 3′ part of MicA (nts 23–74; TMA) to construct RydC-TMA. The 5′ end of RydC is required to interact with cfa mRNA. GFP levels were determined on western blots of total protein samples isolated from Salmonella ΔrydC ΔrpoS mutants carrying plasmids for cfa1::gfp and either a control or plasmids for PL-driven overexpression of TMA, RydC or RydC-TMA. (D) Validation of the RydC-cfa mRNA interaction. Salmonella ΔrydC ΔrpoS mutants carrying plasmids for cfa1::gfp and cfa1*::gfp in combination with a control plasmid or RydC overexpression plasmids pPLRydC and pPLRydC*. Expression of GFP-fusion proteins was monitored on western blots of total protein samples prepared from cells in exponential growth (OD600 of 1). Equal expression of RydC and RydC* was controlled by northern blot analysis. (E) The regulation of cfa by RydC is conserved. GFP expression was monitored by western blot analysis in Salmonella ΔrydC mutant cells carrying translational Salmonella (STM), E. coli (ECO), K. pneumoniae (KPN) or E. aerogenes (EAE) cfa1::gfp reporter fusions in combination with either a control (−) or a plasmid to overexpress RydC versions of the indicated species (+).Source data for this figure is available on the online supplementary information page. Source Data for Figure 4 [embj2013222-sup-0004-SourceData-S4.pdf] Download figure Download PowerPoint Second, computational analysis using the RNAhybrid algorithm (Rehmsmeier et al, 2004) predicted a potential 11-bp RNA duplex to form by the pairing of nucleotides 2–12 of RydC with the region of cfa that is protected in the above structure probing experiments (Figure 4B). With a predicted change in free energy of −22.8 kcal/mol, this RNA duplex was well within the range of previously observed seed pairings (Papenfort et al, 2010). This interaction involved the highly conserved nucleotides of the single-stranded 5′ end of RydC (compare to Figure 1A), reminiscent of the 5′ terminal seed pairing reported for numerous other Hfq-associated sRNAs (Guillier and Gottesman, 2008; Pfeiffer et al, 2009; Papenfort et al, 2010; Corcoran et al, 2012; Holmqvist et al, 2012; Shao et al, 2013). Moreover, a chimaeric RydC-TMA sRNA in which the first 13 nucleotides of RydC are fused to an unrelated sRNA (TMA; truncated MicA; Bouvier et al, 2008) also activated the cfa1::gfp reporter indistinguishably from wild-type RydC, indicating that the 5′ end of RydC is sufficient for recognition of the cfa target (Figure 4C). For further proof that the predicted seed pairing underlies cfa regulation, we changed cytosine −102 to guanosine in the cfa1::gfp reporter. As expected, this mutation to which we refer as cfa1* abolished reporter activation by RydC (Figure 4D, lanes 1–2 versus 5–6). Likewise, mutation of RydC at position 5 from guanosine to cytosine (RydC*) abolished regulation of the wild-type cfa::gfp reporter (Figure 4D, lanes 1–3). However, the combination of these two compensatory mutations restored reporter activation to wild-type levels (Figure 4D, lane 6). The described RydC-cfa seed pairing seemed conserved in many other γ-proteobacterial species, as seen in sequence alignments of RydC sRNA (Figure 1A) or cfa mRNA (Supplementary Figure S6). To test whether the conservation of the interaction sites extends to conservation of regulation, we constructed additional cfa1::gfp reporters with the sequences of E. coli, Enterobacter aerogenes and Klebsiella pneumoniae, and co-expressed each fusion together with RydC of the cognate species. Each of these reporters was found to be activated by the cognate RydC sRNA (Figure 4E). Thus, RydC activates cfa synthesis by a conserved seed pairing interaction with the long isoform of the cfa mRNA, far upstream of the canonical translation control elements. Translation-independent target activation Activation by a bacterial sRNA typically occurs through a targeted disruption of
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