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.

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. Gene expression is regulated at multiple levels, including the decision of whether or not to translate a mRNA. This phenomenon, known as translational regulation, allows rapid changes in cellular concentrations of proteins and is well suited to the adjustment of cellular growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the mRNA to the small ribosomal 30S subunit and so are directly involved in this regulatory process. Structured mRNAs must interact with the 30S subunit in a two-step pathway whereby the docking of a folded mRNA is followed by an accommodation step that involves unfolding of these structures. However, it is not known how the ribosome unfolds mRNA structures to promote translation initiation, nor which ribosomal factors are responsible for this activity. We demonstrate that the first three domains of ribosomal protein S1 endow the 30S subunit with an RNA chaperone activity that is essential for the binding and unfolding of structured mRNAs, allowing the correct positioning of the initiation codon for translation. However, ribosomal protein S1 is not required for all mRNAs and acts differently depending on the type of regulatory elements present in a given mRNA. In all, we have shown that ribosomal protein S1 provides an RNA-melting activity to the exit site of the 30S decoding channel and confers some plasticity on the ribosome to initiate translation of mRNAs.

It is widely recognized that RNA degradation plays a critical role in gene regulation when fast adaptation of cell growth is required to respond to stress and changing environmental conditions. Bacterial ribonucleases acting alone or in concert with various trans-acting regulatory factors are important mediators of RNA degradation. Here, we will give an overview of what is known about ribonucleases in several Gram-positive bacteria, their specificities and mechanisms of action. In addition, we will illustrate how sRNAs act in a coordinated manner with ribonucleases to regulate the turnover of particular mRNA targets, and the complex interplay existing between the ribosome, the ribonucleases and RNAs.

Staphylococcus aureus produces a high number of RNAs for which the functions are poorly understood. Several non-coding RNAs carry a C-rich sequence suggesting that they regulate mRNAs at the post-transcriptional level. We demonstrate that the Sigma B-dependent RsaA RNA represses the synthesis of the global transcriptional regulator MgrA by forming an imperfect duplex with the Shine and Dalgarno sequence and a loop-loop interaction within the coding region of the target mRNA. These two recognition sites are required for translation repression. Consequently, RsaA causes enhanced production of biofilm and a decreased synthesis of capsule formation in several strain backgrounds. These phenotypes led to a decreased protection of S. aureus against opsonophagocytic killing by polymorphonuclear leukocytes compared to the mutant strains lacking RsaA. Mice animal models showed that RsaA attenuates the severity of acute systemic infections and enhances chronic catheter infection. RsaA takes part in a regulatory network that contributes to the complex interactions of S. aureus with the host immune system to moderate invasiveness and favour chronic infections. It is the first example of a conserved small RNA in S. aureus functioning as a virulence suppressor of acute infections. Because S. aureus is essentially a human commensal, we propose that RsaA has been positively selected through evolution to support commensalism and saprophytic interactions with the host. Staphylococcus aureus is a commensal and an opportunistic pathogen that causes a large range of community and hospital-acquired infections. The bacteria produce an array of virulence factors, the expression of which is regulated by a set of regulators including proteins and RNAs. In recent years, a large number of small non-coding RNAs encoded by the S. aureus genome have been identified but determination of their function is still lagging behind. This study shows that RsaA, a staphylococcal conserved non-coding RNA, operates at the post-transcriptional level by repressing the translation of the master regulatory protein MgrA. The repression is based on a direct interaction of RsaA with the ribosome binding site of mgrA mRNA. Through MgrA regulation, RsaA activates biofilm formation and inhibits capsule synthesis. Using appropriate animal models, we showed that RsaA acts as a suppressor of virulence because the deletion of its gene increases the invasiveness of S. aureus in the mice sepsis model. RsaA is thus part of complex regulatory network that modify the interactions of S. aureus with the eukaryotic immune system. These findings illustrate how small RNAs can have a major impact in bacterial biology.

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. Control of mRNA stability is crucial for bacteria to survive and rapidly adapt to environmental changes and stress conditions. The molecular players and the degradation pathways involved in these adaptive processes are poorly understood in Staphylococcus aureus. The universally conserved double-strand-specific endoribonuclease III (RNase III) in S. aureus is known to repress the synthesis of several virulence factors and was recently implicated in genome-wide mRNA processing mediated by antisense transcripts. We present here the first global map of direct RNase III targets in S. aureus. Deep sequencing was used to identify RNAs associated with epitope-tagged wild-type RNase III and two catalytically impaired but binding-competent mutant proteins in vivo. Experimental validation revealed an unexpected variety of structured RNA transcripts as novel RNase III substrates. In addition to rRNA operon maturation, autoregulation, degradation of structured RNAs, and antisense regulation, we propose novel mechanisms by which RNase III increases mRNA translation. Overall, this study shows that RNase III has a broad function in gene regulation of S. aureus. We can now address more specifically the roles of this universally conserved enzyme in gene regulation in response to stress and during host infection.