RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3 domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3 domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3 domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop-loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3 domain in establishing a network of S. aureus virulence factors.[Keywords: Regulatory RNA; translational repression; antisense regulation; RNase III; virulence; Staphylococcus aureus]Supplemental material is available at http://www.genesdev.org.

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism.

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA3cRNA3vRNA), but its transcriptional activity (vRNA3mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNAbinding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.Influenza A virus is a negative-strand RNA virus containing eight segments of single-stranded RNA as its genome (39). The RNA genome is transcribed and replicated by the viral RNA-dependent RNA polymerase in the cell nucleus (21). The viral RNAs (vRNA) are transcribed into mRNAs and replicated through a cRNA intermediate to produce more vRNA molecules. Synthesis of these three RNA species requires different modes of initiation and termination (reviewed in references 23 and 34). Synthesis of mRNAs is primed by short capped RNA fragments that are generated from cellular pre-mRNAs by endonucleolytic cleavage. Consequently, viral mRNA molecules contain a 9-to 17-nucleotide (nt) capped host-derived RNA sequence at their 5Ј ends. On the other hand, the synthesis of cRNA and vRNA molecules is initiated in a primer-independent manner, resulting in triphosphorylated 5Ј ends. Synthesis of mRNAs is prematurely terminated 16 to 17 nucleotides from the 5Ј end of the vRNA template at a sequence of 5 to 7 uridines that acts as a polyadenylation signal (30,47,49). The poly(A) tail is synthesized by the viral RNA polymerase by repeated copying of the U sequence (47). During the synthesis of cRNA molecules, the polyadenylation signal is ignored, resulting in full-length copies of vRNA.All three reactions, i.e., vRNA3mRNA (transcription), vRNA3cRNA (first step of replication), and cRNA3vRNA (second step of replication) are catalyzed b...

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.

This study reveals novel insights into how Escherichia coli ribosomal protein S1 functions as an RNA chaperone on the ribosome, unfolding and positioning mRNAs for translation initiation.

The presence of regulatory sequences in the 3′ untranslated region (3′-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3′-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3′-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3′-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3′-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3′-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3′-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3′-UTR with the 5′-UTR of the same mRNA.

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5P-terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a`transzyme' molecule, the autocatalytic activity of which liberates a 5P-OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNA yr , starting with 5P-C I , and operates as well for yeast tRNA esp with 5P-U I . Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k t and K m parameters quasi identical to those of their phosphorylated counterparts.z 1998 Federation of European Biochemical Societies.

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.