Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae

BACKGROUND: Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45. RESULTS: We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells. CONCLUSIONS: Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors

Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce

A Staphylococcus aureus small RNA is required for bacterial virulence and regulates the expression of an immune-evasion molecule

Staphylococcus aureus, a pathogen responsible for hospital and community-acquired infections, expresses many virulence factors under the control of numerous regulatory systems. Here we show that one of the small pathogenicity island RNAs, named SprD, contributes significantly to causing disease in an animal model of infection. We have identified one of the targets of SprD and our in vivo data demonstrate that SprD negatively regulates the expression of the Sbi immune-evasion molecule, impairing both the adaptive and innate host immune responses. SprD interacts with the 59 part of the sbi mRNA and structural mapping of SprD, its mRNA target, and the ‘SprD-mRNA ’ duplex, in combination with mutational analysis, reveals the molecular details of the regulation. It demonstrates that the accessible SprD central region interacts with the sbi mRNA translational start site. We show by toeprint experiments that SprD prevents translation initiation of sbi mRNA by an antisense mechanism. SprD is a small regulatory RNA required for S. aureus pathogenicity with an identified function, although the mechanism of virulence control by the RNA is yet to be elucidated

Dual RNA regulatory control of a Staphylococcus aureus virulence factor.

International audienceIn pathogens, the accurate programming of virulence gene expression is essential for infection. It is achieved by sophisticated arrays of regulatory proteins and ribonucleic acids (sRNAs), but in many cases their contributions and connections are not yet known. Based on genetic, biochemical and structural evidence, we report that the expression pattern of a Staphylococcus aureus host immune evasion protein is enabled by the collaborative actions of RNAIII and small pathogenicity island RNA D (SprD). Their combined expression profiles during bacterial growth permit early and transient synthesis of Sbi to avoid host immune responses. Together, these two sRNAs use antisense mechanisms to monitor Sbi expression at the translational level. Deletion analysis combined with structural analysis of RNAIII in complex with its novel messenger RNA (mRNA) target indicate that three distant RNAIII domains interact with distinct sites of the sbi mRNA and that two locations are deep in the sbi coding region. Through distinct domains, RNAIII lowers production of two proteins required for avoiding innate host immunity, staphylococcal protein A and Sbi. Toeprints and in vivo mutational analysis reveal a novel regulatory module within RNAIII essential for attenuation of Sbi translation. The sophisticated translational control of mRNA by two differentially expressed sRNAs ensures supervision of host immune escape by a major pathogen

A small RNA controls a protein regulator involved in antibiotic resistance in Staphylococcus aureus.

International audienceThe emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen

Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae.

International audienceBACKGROUND: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3. RESULTS: In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants. CONCLUSION: This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability

Regulatory RNAs Involved in Bacterial Antibiotic Resistance.

International audienceAn increasing number of RNAs have been recently shown to possess regulatory functions similar to those of proteins..

Nothern blotting was used to assess the effect of deletion on the accumulation of mRNA

<p><b>Copyright information:</b></p><p>Taken from "Inactivation of NMD increases viability of nonsense mutants in "</p><p>http://www.biomedcentral.com/1471-2199/8/71</p><p>BMC Molecular Biology 2007;8():71-71.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2039749.</p><p></p> Total RNA was isolated from strain 5B-D1645 () transformed with plasmids pRS316 and pRS316/, designated as () and (), respectively. Northern blots were hybridized with radiolabeled and probes. . Representative hybridization signals specific to mRNA (upper panel), mRNA (middle panel) and actin mRNA () used as an internal control (lower panel) are shown. Numbers indicated under upper and middle panels represent the relative abundance of and mRNA's, respectively, in and strains. (s.d.) – standard deviation. . Accumulation of precursor mRNA was used to control that NMD is altered in the strain. The probe detects both precursor and mature mRNA. The fold increase in precursor/mature mRNA accumulation in strain relative to strain is indicated with the standard deviation (s.d.)