Novel aspects of RNA regulation in Staphylococcus aureus

FEBS Letters xxx (2014) xxx–xxx

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Review

Novel aspects of RNA regulation in Staphylococcus aureus Pierre Fechter, Isabelle Caldelari, Efthimia Lioliou, Pascale Romby ⇑ Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, IBMC, 15 Rue René Descartes, 67084 Strasbourg, France

a r t i c l e

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Article history: Received 30 April 2014 Revised 16 May 2014 Accepted 19 May 2014 Available online xxxx Edited by Wilhelm Just Keywords: Regulatory RNAs Post-transcriptional regulation Virulence Staphylococcus aureus

a b s t r a c t A plethora of RNAs with regulatory functions has been discovered in many non-pathogenic and pathogenic bacteria. In Staphylococcus aureus, recent findings show that a large variety of RNAs control target gene expression by diverse mechanisms and many of them are expressed in response to specific internal or external signals. These RNAs comprise trans-acting RNAs, which regulate gene expression through binding with mRNAs, and cis-acting regulatory regions of mRNAs. Some of them possess multiple functions and encode small but functional peptides. In this review, we will present several examples of RNAs regulating pathogenesis, antibiotic resistance, and host-pathogen interactions and will illustrate how regulatory proteins and RNAs form complex regulatory circuits to express the virulence factors in a dynamic manner. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Multiple roles of bacterial RNAs in gene regulation Besides regulatory transcriptional proteins, RNA molecules are now recognized as key players of gene regulation in all living organisms (e.g. [1]). Mapping of the transcriptional start sites at the genome-wide scale revealed the complexity of the RNA landscape in numerous bacterial genomes, including non-pathogenic and pathogenic bacteria [2]. In bacteria, the small RNAs (sRNAs) often noncoding, accomplish a large variety of regulatory functions and can act at the levels of transcription, translation or RNA degradation. The large majority of them regulate pathways that sense and transfer the external signals, and adapt the cell population in response to stress and environmental changes [3] while others protect the core genome from foreign nucleic acids [4]. Among these sRNAs, many of them regulate gene expression by exploiting their ability to selectively bind to other nucleic acids. In addition, mRNAs also behave as regulatory RNAs or as reservoirs of sRNAs. Although the untranslated regions of mRNAs are in general of small size, mRNAs that encode proteins involved in virulence, stress responses and metabolism carry large 50 or 30 untranslated regions [2]. In these large UTRs, regulatory domains are often embedded that could function as direct sensor of physical cues like RNA thermosensors [5] or of intracellular concentration of metabolites, the so-called riboswitches [6]. Riboswitches exhibit a structured receptor domain specifically recognized by a small metabolite, which provokes premature transcription/anti-termination, translation repression/ ⇑ Corresponding author. Fax: +33 (0)3 88602218. E-mail address: [email protected] (P. Romby).

activation, or cleavage through a conformational switch of the mRNA. In general, riboswitches regulate the downstream mRNAs, which are involved in the uptake or metabolism of the sensed metabolite. Recent works have shown that riboswitches regulate gene expression in non-classical ways in Listeria monocytogenes: two S-adenosylmethionine (SAM) riboswitches function in trans to control the synthesis of the virulence regulator PrfA by binding to the 50 -untranslated region of its mRNA [7] while a B12-riboswitch regulates the transcription of an antisense RNA [8]. Because most of the riboswitches control essential genes and adopt specific binding pockets for small compounds, they have been considered as promising targets for the design of novel antibacterial compounds [9]. The advantageous properties of regulatory RNAs have been recently exploited in synthetic biology to rewire bacterial regulatory circuits [10]. In this short review, we will illustrate how several regulatory RNAs in Staphylococcus aureus fine tune the expression of key transcriptional factors and how they are embedded in complex regulatory circuits, which link virulence to stress adaptation and metabolism. For more detailed information, we refer to the recent reviews on S. aureus regulatory RNAs [11–14]. 2. The quorum sensing dependent RNAIII, a multifaceted regulatory RNA S. aureus is able to adapt to a wide variety of ecological niches. It is a commensal bacterium of skin and anterior nares of a large proportion of the human population but is responsible for numerous hospital- and community-acquired infections (e.g. [15]). This

http://dx.doi.org/10.1016/j.febslet.2014.05.037 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Fechter, P., et al. Novel aspects of RNA regulation in Staphylococcus aureus. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.05.037

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opportunistic pathogen combats the host immune system by producing a battery of virulence factors that are responsible for adhesion, invasion and dissemination in host tissues and for acquisition of nutrients [16,17]. Expression of these factors is temporally regulated by multiple regulators involving two component systems, transcriptional regulatory proteins, and RNAs [18]. Among these systems, R. Novick and coworkers have identified two intracellular effectors of the accessory gene regulator (agr) quorum sensing system, the sensor protein AgrA and the regulatory RNAIII (Fig. 1A), which are both pivotal in S. aureus virulence [16,17,19]. The agr system is composed of two divergent transcripts, RNAII that encodes a quorum sensing cassette (AgrBD) and a two-component system (AgrAC) and RNAIII, a multifunctional RNA (Fig. 1A). The quorum sensing cassette produces an autoinducer peptide (AgrD), which upon a threshold concentration, activates the membrane kinase AgrC and the response regulator AgrA through a cascade of phosphorylation reactions. In turn, AgrA activates the synthesis of its own operon (RNAII) and of RNAIII, but also up-regulates the phenol-soluble-modulin (PSM) cytolysin genes, and down-regulates genes involved in carbohydrate and amino acid metabolism [19]. The bifunctional RNAIII is both a mRNA encoding the PSM d-hemolysin (hld), and a regulator which promotes the switch between the expression of surface proteins and the synthesis of excreted toxins ([17]; Fig. 1A). It is still not known if the translation of hld may alter the regulatory activities of RNAIII but the presence of an open reading frame within RNAIII is not found in all staphylococcal strains [20]. Intriguingly, the translation of hld was delayed

by 1 h compared to RNAIII synthesis, and was abolished by the deletion of its 30 UTR [21]. These data suggested the involvement of a trans-acting factor and/or a conformational change to induce hld translation. The non-coding parts of RNAIII are the regulatory domains: its 50 UTR binds to the leader region of hla mRNA encoding a-hemolysin to facilitate ribosome recruitment while its large 30 UTR is primarily acting as a repressor domain. The 30 UTR is the most highly conserved domain of RNAIII. It is characterized by several C-rich sequence motifs located in unpaired regions, which represent the seed sequences to promote basepairing interactions with target mRNAs (Fig. 1A). Although the topologies of the repressed mRNA–RNAIII complexes vary, binding of RNAIII prevents ribosome binding in all cases, which is usually followed by the rapid degradation of the repressed mRNAs [22–24]. These mRNAs encode virulence factors expressed at the surface of the cell (protein A, coagulase, SA1000), and the transcriptional repressor of toxins, Rot. A recent work has shown that three distant regions of RNAIII including its 50 and 30 UTRs bind respectively to the coding region and the ribosome binding site of sbi mRNA to prevent its translation ([25,26]; Fig. 1A). Although distant domains of RNAIII bind to target mRNAs (Fig. 1A), only the region encompassing H13 and H14 of RNAIII is essential for translation repression while the other domains of RNAIII reinforce the stability of the RNAIII– mRNA complexes [24,25]. In addition to protein A, Sbi protein is another immunoglobulin-binding cell surface protein that protects the bacteria from the host immune system [27]. Hence, RNAIII decreases the production of two major adhesins, proteins A and

Fig. 1. Examples of regulatory RNAs and their mechanism of action. (A) Genomic organization of the quorum sensing agr system and mechanisms of repression by RNAIII on specific target mRNAs. The schematic secondary structure of RNAIII is given. The hld gene encoding d-hemolysin is in green. The various C-rich sequence motifs of RNAIII (in red) represent the seed sequences that bind to the Shine and Dalgarno (SD) sequence of mRNA targets. Various topologies of inhibitory RNAIII-mRNA are given. These topologies prevent binding of the 30S small ribosomal subunit (30S) and for several mRNAs, the duplexes are appropriate to promote specific cleavage by the double strand specific RNase III (grey arrows). The data for spa, rot, and sbi regulations are from Huntzinger et al. [22], Boisset et al. [24] and Chabelskaya et al. [36], respectively. (B) Repression of sbi mRNA translation by SprD. Three different regions of SprD form basepairing interactions with the 50 untranslated region and the beginning of the coding sequence of sbi mRNA. The duplex prevents the formation of the initiation ribosomal complex. The data are from Chabelskaya et al. [25]. (C) Repression of mgrA mRNA by the small non coding RNA, RsaA. Two regions of RsaA form a duplex with the ribosome loading site stabilized by a loop-loop interaction within the coding region. The two regions are essential to repress translation, which is subsequently followed by the degradation of the mRNA. The data are adapted from Romilly et al. [46]. T is for Rho-independent terminator of transcription.

Please cite this article in press as: Fechter, P., et al. Novel aspects of RNA regulation in Staphylococcus aureus. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.05.037

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Fig. 2. Regulatory circuits involved in virulence gene expression. The networks are based on the knowledge acquired from the literature (see the accompanying text). The transcriptional regulatory proteins are in black, the regulatory RNAs are in red and the target proteins are in purple. The transcriptional regulation is shown by black lines while post-transcriptional regulation is shown by red lines. Arrows corresponded to activation while bars corresponded to repression. Indirect regulation is given by dotted lines. The functional consequences of the regulation are also given.

Sbi, required to escape the innate host immunity. In contrast, RNAIII indirectly activates the transcription of exotoxins through the inhibition of Rot [24,28]. Thus, these regulatory circuits ensure tight regulation and low noise when RNAIII production dominates (for a review, [29]). They also create a delay between the repression of adhesin synthesis and the induction of exotoxin production enabling an effective transition of the pathogen for spreading and dissemination in host tissues, and establishment of the infection following quorum sensing signal ([17]; Fig. 2). High production of RNAIII is expected to limit the cell-to-cell variability of gene expression that might be important in establishing the cellular phenotype. The action of RNAIII is not restricted only to virulence gene regulation. Indeed, its conserved 30 end domain also represses at the post-transcriptional level the synthesis of several cell wall hydrolytic enzymes ([24,30]; Lioliou et al., unpublished data). These enzymes have been associated with numerous processes such as peptidoglycan maturation and turnover, cell division and separation, protein secretion, biofilm formation, and programmed cell death. Hence, we propose that the RNAIII-dependent inhibition may contribute to prevent accidental cell lysis at high cell density, and to maintain the cell wall properties for the adaptation of the bacteria to the host and to environmental conditions. 3. Crosstalk between regulatory RNAs and the quorum sensing system Although the importance of the agr system in pathogenesis is well recognized, the expression of RNAIII varies considerably in clinical isolates from acute infections [31]. It is thus of importance to understand how variation in the concentration of the agr encoded proteins and RNAIII may affect the regulatory circuits

and what would be the functional consequences for S. aureus pathogenicity. Numerous works have demonstrated that the agr operon is controlled at the transcriptional level by several global transcriptional factors like SigB, SarA, and MgrA ([18]; Fig. 2) but also at the post-transcriptional level. Unexpectedly, the CshA RNA helicase has been shown to modulate the stability of agr polycistronic mRNA [32]. One can argue that controlling agr mRNA stability is particularly well appropriate to turn off rapidly the response of cells that are released from a dense population. Under such conditions, the bacteria would be able to rapidly restore the expression of surface proteins required for adhesion and protection against the host immune system. Therefore controlling the agr system independently of the quorum sensing signal might confer to the bacteria the advantage to respond to multiple external signals or environmental cues [18] and to produce the virulence factors at the appropriate moment and place. Recently, the functional characterization of several RNAs in S. aureus has exploded. Based on these findings, we will illustrate here how short and long RNAs crosstalk with the quorum sensing system to regulate the expression of virulence factors (Figs. 1 and 2). Most of these RNAs regulate gene expression through the formation of basepairing interactions with mRNA targets and contribute to mixed regulatory circuits with transcriptional regulatory proteins (Fig. 2). What could be the advantages for the bacteria to evolve these circuits involving RNAs and proteins? Computational studies have shown that RNA-based regulation is sensitive, provides a fast response, and strongly represses variations in protein expression, which are characteristics particularly suitable for stress responses [29,33,34]. The effectiveness of the regulation by RNAs targeting mRNAs depend both essentially on the ratio of the regulatory RNA and the mRNA production rates and on the binding rates. As described for RNAIII, the regulatory RNA usually serves as a node

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in a regulatory cascade and transcriptional and post-transcriptional controls are combined resulting in tight repression. 3.1. A virulence factor regulated by RNAIII and the pathogenicity island encoded SprD ncRNA In addition to the core genome, mobile genetic elements such as pathogenicity islands and transposons also express sRNAs [35]. One of these sRNAs, SprD was shown to crosstalk with the core genome by repressing the translation of sbi mRNA [25,36]. At the early exponential phase when the levels of SprD are high, it binds to the ribosome binding site of sbi mRNA to prevent the formation of the initiation ribosomal complex (Fig. 1B). At high cell density, SprD decreases and concomitantly RNAIII synthesis accumulates [25]. Under these conditions, RNAIII takes the relay of SprD to repress the translation of sbi mRNA using a similar mechanism, i.e. by blocking the access of the ribosome to initiate translation. As for many of its mRNA targets, the 30 domain of RNAIII was sufficient for sbi repression because this 30 domain forms basepairing interactions with the ribosome binding site of sbi. Based on the differential expression of the two RNAs, it was proposed that the action of RNAIII and SprD restrained the synthesis of Sbi during a short specific window of the growth. The use of several RNAs to control the expression of one mRNA is not restricted to S. aureus. This has been well described in enterobacteriaceae where two sRNAs coordinate the repression of several outer membrane proteins in a parallel but independent manner [37]. 3.2. AgrA represses a toxin-regulating non-coding RNA, ArtR Besides RNAIII, a recent study revealed the existence of a second RNA that indirectly activates the synthesis of a-hemolysin [38]. This RNA, called AgrA-repressed toxin-regulating sRNA (ArtR), is repressed at high cell density by AgrA (Fig. 2). In addition, ArtR is able to form basepairing interactions with the 50 UTR of sarT mRNA leading to rapid degradation mediated by the double-strand specific RNase III. SarT belongs to the DNA binding protein family homologous to SarA, a staphylococcal accessory regulator of virulence gene expression in S. aureus [39]. SarT represses the transcription of hla mRNA encoding a-hemolysin [40] suggesting that ArtR acts indirectly on the exotoxin production. It is intriguing that ArtR transcription is repressed by AgrA suggesting that ArtRmediated hla up-regulation would play a predominant role in agr-deficient strains or in strains expressing low agr levels. Interestingly the 30 end of ArtR overlaps with the 30 end of luxS mRNA indicating that ArtR is also acting as an antisense RNA. LuxS/AI-2 is part of a signaling pathway in S. aureus, which regulates biofilm formation in an IcaR-dependent manner and capsular polysaccharide production via the two-component system KdpDE [41]. Interestingly, it has been shown that the luxS and the agr systems have cumulative effects on biofilm formation [42]. It is thus tempting to propose that the regulation of ArtR transcription might have some consequences on the signaling pathways mediated by luxS. Therefore, this study suggested that ArtR could act on the same locus as a fully complementary antisense RNA, or at a distant gene locus through imperfect interactions. 3.3. Acquisition of a novel RNA caused perturbation of the agr system The acquisition of multi-resistance toward antibiotics is one of the major problems to treat S. aureus infections. Community and hospital acquired-methicillin resistant S. aureus (CA-, and HAMRSA, respectively) cause severe infections in healthy patients. CA-MRSA are usually more susceptible to anti-microbial drugs but are more virulent than HA-MRSA. The antibiotic resistance gene mecA is encoded on a pathogenicity island, the so-called

staphylococcal cassette chromosome mec (SCC-mec). A recent study revealed that SCCmec contains additional genes beyond mecA, which includes psm-mec encoding a small cytolytic toxin [43]. The psm-mec gene is absent or not expressed in many CAMRSA questioning its importance in virulence. Functional analysis revealed that psm-mec RNA transcript is functional and acts as an antisense RNA to repress the translation of agrA mRNA [43]. Binding of the regulatory RNA occurred in the coding sequence of agrA mRNA suggesting that the regulation would affect the stability of the mRNA (Fig. 2). Deletion of the psm-mec gene from HA-MRSA increased the yield of AgrA and the mutated strains were more virulent in mice infection model. Therefore, it was suggested that decreased levels of one of the quorum sensing intracellular effectors reduce virulence in HA-MRSA. Horizontal genetic transfer by mobile genetic elements plays an important role in the evolution of S. aureus strains. This study illustrates how the acquisition of a novel regulatory RNA is able to interfere with the quorum sensing system. 3.4. A non-coding RNA as a repressor of capsule formation Staphylococcal capsule polysaccharides are important in the pathogenesis of S. aureus infections. This structure protects S. aureus against the host immune system, and prevents opsonophagocytic killing by leukocytes resulting in bacterial persistence in the bloodstream of the host. The expression of the capsule appears to be predominant in isolates from patients with acute infection [44]. The formation of capsule is strongly regulated and one of the major regulators in vivo is MgrA, another SarA homolog [45]. Recent findings showed that the sRNA RsaA represses the synthesis of the global transcriptional activator MgrA by an antisense mechanism (Fig. 1C). RsaA binds to two distant regions of mgrA mRNA, and via its C-rich sequence motif interacts with the SD sequence to repress translation initiation [46]. Phenotypic analysis revealed that RsaA enhances biofilm formation and conversely, decreases synthesis of capsule formation, and favors opsonophagocytic killing of S. aureus by polymorphonuclear leukocytes. Consistently, in vivo, the expression of RsaA contributed to attenuate the severity of systemic infections and to enhance chronic infection, suggesting that RsaA functions as a virulence suppressor of acute infections. In addition, the synthesis of RsaA is under the control of the pleiotropic transcriptional regulator SigB [47]. SigB enhances resistance to oxidative and UV stresses, modulates biofilm and capsule formation, and regulates the transcription of virulence genes in an opposite manner to RNAIII [48]. Moreover, MgrA is activated by the two component system arlRS, whose expression is controlled by SigB [49] showing that the expression of MgrA is under the control of an incoherent feed-forward loop, involving a positive loop regulated by ArlRS and a negative loop under the control of RsaA (Fig. 2). In addition, MgrA activates the agr system and enhances RNAIII levels [50,51]. Therefore, RsaA functionally links the global regulators SigB and MgrA, with possible results on the temporal expression of the virulence determinants independently of the quorum sensing signal (Fig. 2). Finally, RsaA and a second sRNA SprX, repress the synthesis of SpoVG, required for capsule synthesis and bacterial resistance to methicillin and glycopeptides [26]. Whether the two RNAs act synergistically or independently remains to be studied. In contrast to SprX, RsaA is expressed in all staphylococci [46,47] suggesting that RsaA has been most likely selected to promote saprophytic interactions with the host. 4. Widespread antisense regulation involving mRNAs Post-transcriptional regulation is often used to modulate gene expression in a wide range of biological functions particularly when fast adaptation is required. Compared to transcriptional

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regulation, it has the obvious advantages to provide a more rapid response, to fine-tune the yield of protein synthesis, and provide to the cells many more possibilities to sense and respond to external or internal signals. In these mechanisms, the structures of the mRNAs actively contribute to these regulations. Because the rate limiting step of translation, the initiation process, is one of the key step for control, many studies have been focused on the 50 UTR of mRNAs [52]. The 50 UTRs of S. aureus mRNAs often carry secondary structures that can perturb the transcription– translation synchronization with direct consequences on premature transcription termination, translation initiation and mRNA processing, and degradation. These structures offer specific binding sites for trans-acting factors such as small metabolites (riboswitches), RNA-binding proteins, ribonucleases, and sRNAs [11]. We have illustrated above with specific examples that the SD sequence of mRNAs is often the target of sRNAs, which contain C-rich sequence motifs to prevent the ribosome recruitment. A precise mapping of the 30 ends of well-annotated mRNAs was recently done using both RNA-seq and tiling arrays [8]. This work showed that large 30 UTRs (>100 nt) of mRNAs have been underestimated and are frequently found in S. aureus [53,54], suggesting that they can potentially host regulatory elements (Fig. 3A–D). Strikingly, transcription readthrough at the Rho-independent terminator of mRNAs has been often observed generating large 30 UTRs, which overlap with mRNAs encoded on the opposite genomic strand (Fig. 3B). Thus, these mRNAs can also act as antisense RNA to inhibit the neighboring mRNA or operons, contributing to fine-tune gene expression [12,55]. A key enzyme, the double strand specific RNase III, is associated with this antisense regulation to cleave the long duplexes ([55,56]; Fig. 3A). Besides short antisense

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RNAs have also been identified. For instance, a recent work has evidenced a novel type I toxin–antitoxin system in S. aureus encoded from pathogenicity islands [57]. The short antisense RNA SprF1 binds to the 30 UTR of the ORFs encoded by SprG1 and induces its rapid degradation most probably by RNase III ([56]; Fig. 3A). Unexpectedly, SprG1 produces two peptides from two in-frame initiation codons, which are able to lyse host cells as well as other bacteria. Whether these two peptides are differentially expressed during cell growth, colonization and/or infection remains unanswered [57]. Although the function of this SprG1–SprF1 module in virulence is not proven, it is well known that type I toxin– antitoxin systems slow down growth in response to stress, and can contribute to formation of persister cells that resist to antibiotic treatment [58,59]. The presence of divergent mRNAs presenting overlaps with either their entire length or over their 50 or 30 -untranslated regions seems to be more general rather than an exception in the S. aureus genome [53,55], and has also been observed in L. monocytogenes [53]. Among this class of long asRNA, a novel type of genomic locus, called excludon has been described in L. monocytogenes; this organization comprises a long asRNA that covers divergent genes or operons involved in the same pathways but with opposite functions, allowing regulatory switches in response to a specific signal [60,61]. Another class of long 30 UTRs includes riboswitch sequences [53]. Most of the riboswitches in S. aureus regulate termination of transcription and respond to the intracellular concentration of S-adenosyl-methionine, thiaminepyrophosphate, lysine, glycine, pre-Q, flavine mononucleotide, and guanine [62]. When the Rho-independent terminator is formed as the result of the metabolite binding, it prevents the synthesis of the downstream

Fig. 3. Novel examples of regulation involving the 30 untranslated (UTR) regions of mRNAs. (A) An example of type I toxin–antitoxin system. The short antisense RNA (SprF1) represses the synthesis of two cytolytic peptides encoded by SprG1. The interaction involves fully complementary basepairings with the 30 UTR of SprG1 and probably results in the rapid degradation of SprG1 [57]. (B) Transcription readthrough to generate long 30 UTRs. In several examples, transcription of the mRNA continues despite the presence of a Rho-independent terminator close to the stop codon [53]. This generates a long 30 UTR, which overlaps with the divergent mRNA. The basepairing interactions are then processed by RNase III [55]. A characteristic example is given for SA0367 gene encoding a NADPH flavin oxidoreductase protein, and SA0368 encoding a protein similar to a sodium dicarboxylate symporter protein. (C) Several RNA transcripts stop at a Rho-independent transcription terminator that is part of a riboswitch [53]. When the metabolite (black circle) binds to the riboswitch as shown by the secondary structure, it stabilizes the formation of a terminator (T). As a consequence, the transcription of the downstream gene is arrested while the upstream gene generates a mRNA with a long 30 UTR containing the riboswitch. At low concentration of the metabolite, an antiterminator structure is formed allowing transcription, and a long polycistronic RNA is generated. A typical example is given for the glycine-sensing riboswitch. (D) The icaR mRNA encodes a transcriptional repressor of biofilm. The mRNA carries a long 30 UTR which hinders the ribosome binding in the 50 UTR to weaken the translation efficiency of icaR mRNA, and to induce its degradation by RNase III [53]. An interaction between the C-rich sequence motif and the Shine and Dalgarno (SD) sequence has been demonstrated [53]. As a result, the icaADBC operon is made to synthesize the exopolysaccharide and to promote the biofilm development. T is for Rho-independent terminator of transcription.

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gene but in addition serves as a terminator for the upstream gene (Fig. 3C). Recent findings revealed the importance of a long 30 UTR in icaR mRNA, which encodes a transcriptional repressor of biofilm formation. The ability of S. aureus to form biofilms protects the bacteria against host defenses, antibiotic therapies, and is one major cause of hospital-acquired infections [63]. The main exopolysaccharidic polymer PIA-PNAG constituting the biofilm matrix depends on the operon icaADBC, which is controlled by the transcriptional regulatory protein IcaR. The 390 nt-long 30 UTR of icaR is a negative determinant that interferes with the translation initiation of its own mRNA (Fig. 3D). The 30 UTR contains a C-rich sequence motif that is required for binding to the SD sequence to prevent ribosome binding, and to recruit RNase III for cleavage [53]. Disrupting this interaction resulted in the accumulation of IcaR and inhibited biofilm formation. This study shows that the 30 UTR can block the ribosome binding site in the 50 UTR by a mechanism that is reminiscent of RNAIII and other trans-acting sRNAs from S. aureus (Fig. 3D). Whether basepairings between the 30 and 50 UTRs occur on the same icaR mRNA or between two mRNA molecules was not yet demonstrated. As transcription is coupled to translation, access of the ribosome on its loading site has to be prevented before the 30 UTR is made. Analysis of the 50 UTR structure suggested that the SD sequence is sequestered into a hairpin motif that would impair efficient recognition by the ribosome but is sufficiently unstable to be displaced by the 30 UTR binding [53]. Therefore, the 5’UTR undergoes a conformational rearrangement mediated by the 30 UTR to irreversibly block icaR translation. It remains to be analyzed if specific trans-acting factors would contribute to relieve the 50 –30 UTR pairings to promote the synthesis of IcaR. Given the fact that many mRNAs carry large 30 UTRs, novel regulatory mechanisms are expected to be discovered. For instance, these regions might contain specific binding sites for trans-acting factors modifying the mRNA stability, or can also be the reservoir of small regulatory RNAs [64]. 5. Conclusion With the new developments of genome- and proteome-wide approaches, the functions of S. aureus sRNAs in gene regulation, and their impact on virulence can now be better apprehended. Finding their primary and secondary targets is still a difficult but essential task to gain knowledge on the strategies that S. aureus has evolved to respond to environment cues and to the host. Furthermore, more details should be obtained on the contribution of RNA-binding proteins and ribonucleases in the sRNA functioning and regulation. Global approaches performed on SarA [65], the RNA helicase CshA [32] and several ribonucleases [55,56,66,67] strongly suggest that these proteins reshape the gene expression pattern at the post-transcriptional level. Whether specific machinery is associated with the sRNA-dependent regulation needs to be clarified. Several studies have shown how sRNAs expressed from mobile elements, which encode for virulence factors and resistance gene, affected the translational regulation of genes of the core genome. Understanding the relationships between mobile genetic elements and the core genome, as well as the impact of the acquisition of gene resistance on the physiopathology should give some insights on the evolution of S. aureus strains. A recent study has shown that the expression of the erythromycin resistance genes reduced cell fitness due to modification of nucleotides of the 23S rRNA in the ribosomal tunnel, which deregulate the expression of a set of proteins [61]. Hence, the resistance genes permit to the cells to survive upon antibiotic treatment, but their acquisition also affected the physiology of the cell. With the new development of dual RNA-seq [68] and ribosome profiling [69], it

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Please cite this article in press as: Fechter, P., et al. Novel aspects of RNA regulation in Staphylococcus aureus. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.05.037