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Staphylococcus aureus RsaG is a 3′-untranslated region (3′UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5′- to 3′-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5′-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3′UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3′UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches., This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the French National Research Agency ANR (ANR-18-CE12-0025-04 CoNoCo to P.R.). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021–2028 program of the University of Strasbourg, CNRS, and Inserm was supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US (ANR 20-SFRI-0012), and by EUR IMCBio (IMCBio ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. ED and LB were supported by the “Fondation pour la Recherche Médicale” (FDT201904007957 and ECO202006011534)

SummaryEscherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.

Staphylococcus aureus est une bactérie commensale retrouvée chez environ 30 % des individus sains dont elle colonise la peau et la muqueuse nasale. Cependant, c’est également une bactérie pathogène opportuniste responsable d’infections diverses telles que orgelet, ostéomyélite, endocardite, ou encore septicémie en envahissant un grand nombre de tissus et d’organes. Cette bactérie est capable de s’adapter à des conditions hostiles et variées, telles que carence nutritive et stress osmotique, oxydant, ou thermique, ainsi qu’à la réponse immunitaire de l’hôte, car elle produit une grande diversité de facteurs de virulence. La synthèse de ces facteurs est finement régulée par des protéines et des ARN régulateurs majoritairement non codants, souvent désignés par l’abréviation sARN (dérivée de l’anglais, small RNA). Les facteurs de transcription et les systèmes à deux composants contrôlent l’expression des gènes impliqués non seulement dans le métabolisme, mais aussi dans la réponse au stress et la virulence [1]. Par exemple, la protéine du contrôle catabolique (carbon catabolite control protein A, CcpA) a un rôle essentiel dans le choix de la source carbonée en régulant le métabolisme central de la bactérie ainsi que la virulence [2, 3]. CcpA se fixe à une séquence promotrice spécifique appelée cre (catabolite-responsive element), qui est très conservée chez les bactéries à Gram positif [2]. Quant aux sARN, ils interagissent principalement avec leurs ARN messagers (ARNm) cibles. L’hybridation peut conduire à la stabilisation/ déstabilisation de l’ARNm ou à l’activation/répression de sa traduction [4]. Nous avons montré que la transcription du sARN RsaI (RNA Staphylococcus aureus I) est réprimée par CcpA en présence de glucose [5]. L’induction de la synthèse de RsaI signale que la concentration en glucose diminue dans le milieu extracellulaire et que la croissance des bactéries est ralentie. En interagissant avec ses ARNm cibles ou d’autres sARN, il permet à la population bactérienne de modifier son métabolisme lorsque la source carbonée primaire est consommée.

Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.

RNA modifications are involved in numerous biological processes. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base-pairing formation, protein recognition, RNA structure and stability. tRNAs are the most abundantly modified RNA molecules. Analysis of the roles of their modifications in response to stress, environmental changes, and infections caused by pathogens, has fueled new research areas. Nevertheless, the detection of modified nucleotides in RNAs is still a challenging task. We present here a reliable method to identify and localize tRNA modifications, which was applied to the human pathogenic bacteria, Staphyloccocus aureus. The method is based on a separation of tRNA species on a two-dimensional polyacrylamide gel electrophoresis followed by nano liquid chromatography-mass spectrometry. We provided a list of modifications mapped on 25 out of the 40 tRNA species (one isoacceptor for each amino acid). This method can be easily used to monitor the dynamics of tRNA modifications in S. aureus in response to stress adaptation and during infection of the host, a relatively unexplored field.

Pathogenic bacteria must rapidly adapt to ever‐changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA‐repressed small non‐coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3′ untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose‐6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested., This work was supported by the Centre National de la Recherche Scientifique (CNRS) to [P.R.] and by the Agence Nationale de la Recherche (ANR, grant ANR‐16‐CE11‐0007‐01, RIBOSTAPH, to [P.R.]). It has also been published under the framework of the LABEX: ANR‐10‐LABX‐0036 NETRNA to [P.R.], a funding from the state managed by the French National Research Agency as part of the investments for the future program. The work is financed by a “Projet international de coopération scientifique” (PICS) No. PICS07507 between France and Spain to [I.C.]. D. Bronesky was supported by Fondation pour la Recherche Médicale (FDT20160435025). A. T‐A is financed by the Spanish Ministry of Economy and Competitiveness (BFU2014‐56698‐P) and the European Research Council Consolidator Grant (646869‐ReguloBac‐3UTR).

Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals or nutrient availability resulting in metabolism remodeling. The carbon catabolite repression represents a global regulatory system, allowing the bacteria to express genes involved in carbon utilization and metabolization of the preferred carbon source. InStaphylococcus aureus, regulation of catabolite repressing genes is mediated by the carbon catabolite protein A (CcpA). Here, we have identified a CcpA-dependent small non-coding RNA, RsaI that is inhibited by high glucose concentrations. RsaI represses the translation of mRNAs encoding a major permease of glucose uptake, the FN3K enzyme that protects proteins against damages caused by high glucose concentrations, and IcaR, the transcriptional repressor of exopolysaccharide production. Besides, RsaI regulates the activities of other sRNAs responding to the uptake of glucose-6 phosphate or NO. Finally, RsaI inhibits the expression of several enzymes involved in carbon catabolism pathway, and activates genes involved in energy production, fermentation and NO detoxification when the glucose concentration decreases. This multifunctional RNA provides a signature for a metabolic switch when glucose is scarce and growth is arrested.

International audience; RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases. This method has proven its efficiency to study in vitro the organization of stable RNA-protein complexes. Nevertheless, when the protein binds the RNA very dynamically, with high off-rates, protections are very often difficult to observe. For the analysis of these transient complexes, we describe an alternative strategy adapted from the Site Directed Chemical Probing (SDCP) approach and we compare it with classical footprinting. SDCP relies on the modification of the RNA binding protein to tether an RNA probe (usually Fe-EDTA) to specific protein positions. Local cleavages on the regions of interaction can be used to localize the protein and position its domains on the RNA molecule. This method has been used in the past to monitor stable complexes; we provide here a detailed protocol and a practical example of its application to the study of Escherichia coli RNA chaperone protein S1 and its transitory complexes with mRNAs.

Chemical probing is often used to gain knowledge on the secondary and tertiary structures of RNA molecules either free or engaged in complexes with ligands. The method monitors the reactivity of each nucleotide towards chemicals of various specificities reflecting the hydrogen bonding environment of each nucleotide within the RNA molecule. In addition, information can be obtained on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or perturbation of the environmental cues. The detection of the modifications can be obtained either by using end-labeled RNA molecules or by primer extension using reverse transcriptase. The goal of this chapter is to provide the reader with an experimental guide to probe the structure of RNA in vitro and in vivo with the most suitable chemical probes.

Chemical probing is often used to gain knowledge on the secondary and tertiary structures of RNA molecules either free or engaged in complexes with ligands. The method monitors the reactivity of each nucleotide towards chemicals of various specificities reflecting the hydrogen bonding environment of each nucleotide within the RNA molecule. In addition, information can be obtained on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or perturbation of the environmental cues. The detection of the modifications can be obtained either by using end-labeled RNA molecules or by primer extension using reverse transcriptase. The goal of this chapter is to provide the reader with an experimental guide to probe the structure of RNA in vitro and in vivo with the most suitable chemical probes.

Summary Small non-coding RNA molecules (sRNA) are key regulators participating in complex networks, which adapt metabolism in response to environmental changes. In this issue of Molecular Microbiology, and in a related paper in Proc. Natl. Acad. Sci. USA, Moreno et al. (2011) and Sonnleitner et al. (2009) report on novel sRNAs, which act as decoys to inhibit the activity of the master post-transcriptional regulatory protein Crc. Crc is a key protein involved in carbon catabolite repression that optimizes metabolism improving the adaptation of the bacteria to their diverse habitats. Crc is a novel RNA-binding protein that regulates translation of multiple target mRNAs. Two regulatory sRNAs in Pseudomonas putida mimic the natural mRNA targets of Crc and counteract the action of Crc by sequestrating the protein when catabolite repression is absent. Crc trapping by a sRNA is a mechanism reminiscent to the regulation of the repressor of secondary metabolites (RsmA) in Pseudomonas, and highlights the suitability of RNA-dependent regulation to rapidly adjust cell growth in response to environmental changes.

International audience; Staphylococcus aureus RNAIII is one of the main intracellular effectors of the quorum-sensing system. It is a multifunctional RNA that encodes a small peptide, and its noncoding parts act as antisense RNAs to regulate the translation and/or the stability of mRNAs encoding transcriptional regulators, major virulence factors, and cell wall metabolism enzymes. In this review, we explain how regulatory proteins and RNAIII are embedded in complex regulatory circuits to express virulence factors in a dynamic and timely manner in response to stress and environmental and metabolic changes.

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.

International audience; During the past few years, our knowledge on RNA-based regulation in many organisms has tremendously increased. In bacteria, although transcriptional regulatory proteins remain key players in gene regulation, a wide variety of post-transcriptional regulatory mechanisms discovered highlights the importance of the mRNA structure in the regulation of gene expression. RNA-dependent regulation largely contributes to rapidly adapt the bacterial metabolism in response to environmental changes, stress and in establishment of virulence. Bacteria exploit the extraordinary ability of mRNA to fold into different structures in response to various signals (environmental cues, ligand binding). Induced mRNA conformational rearrangements can potentially regulate transcription, translation and mRNA stability. The present review focuses on the structures of regulatory regions of mRNA that have evolved to permit productive interactions with trans-acting regulators, such as protein or non-coding RNAs. Finally, we describe how particular properties of these regulatory complexes regulate translation initiation.

International audience; Cold induction of cspA, the paradigm Escherichia coli cold-shock gene, is mainly subject to posttranscriptional control, partly promoted by cis-acting elements of its transcript, whose secondary structure at 37 degrees C and at cold-shock temperature has been elucidated here by enzymatic and chemical probing. The structures, which were also validated by mutagenesis, demonstrate that cspA mRNA undergoes a temperature-dependent structural rearrangement, likely resulting from stabilization in the cold of an otherwise thermodynamically unstable folding intermediate. At low temperature, the "cold-shock" structure is more efficiently translated and somewhat less susceptible to degradation than the 37 degrees C structure. Overall, our data shed light on a molecular mechanism at the basis of the cold-shock response, indicating that cspA mRNA is able to sense temperature downshifts, adopting functionally distinct structures at different temperatures, even without the aid of trans-acting factors. Unlike with other previously studied RNA thermometers, these structural rearrangements do not result from melting of hairpin structures.

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International audience; Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

International audience; Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.

Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.