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Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

Chevalier C, Boisset S, Romilly C, Masquida B, Fechter P, Geissmann T, Vandenesch F, Romby P - PLoS Pathog. (2010)

Bottom Line: 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.It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, Strasbourg, France.

ABSTRACT
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.

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RNAIII binds efficiently to coa mRNA in vitro.(A) Determination of the apparent dissociation constant for RNAIII-coa mRNA complex. 5′-end-labeled coa mRNA was incubated alone (−) or with various concentrations of unlabeled wild type RNAIII, the 3′ domain, RNAIII-Δ7-9, and RNAIII-Δ13 (1, 5, 10, 20, 50, 100, 200, and 250 nM). The fraction of labeled coa mRNA associated with RNAIII or its derivatives was calculated from the counts in the corresponding band relative to the total counts in the lane. The Kd value was estimated as the concentration of RNAIII allowing 50% of coa mRNA binding. (B) Binding rate constant for various RNAIII-coa mRNA complexes as determined from three independent experiments. 5′-end-labeled coa mRNA (0.1 nM) was incubated with unlabeled RNAIII (20 nM), RNAIII-Δ7-9 (20 nM), and RNAIII-Δ13 (20 nM) at 37°C. Aliquots were withdrawn at various times (from 0 to 350 sec). The percentage of free coa mRNA was plotted as a function of time to estimate the association rate constant according to [40]. The values for the binding rate constants are the means of three independent experiments: 1.1×105 M−1 s−1 (RNAIII), 9.5×105 M−1 s−1 (RNAIII-Δ7-9), and 1.1×104 M−1 s−1 (RNAIII-Δ13).
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ppat-1000809-g004: RNAIII binds efficiently to coa mRNA in vitro.(A) Determination of the apparent dissociation constant for RNAIII-coa mRNA complex. 5′-end-labeled coa mRNA was incubated alone (−) or with various concentrations of unlabeled wild type RNAIII, the 3′ domain, RNAIII-Δ7-9, and RNAIII-Δ13 (1, 5, 10, 20, 50, 100, 200, and 250 nM). The fraction of labeled coa mRNA associated with RNAIII or its derivatives was calculated from the counts in the corresponding band relative to the total counts in the lane. The Kd value was estimated as the concentration of RNAIII allowing 50% of coa mRNA binding. (B) Binding rate constant for various RNAIII-coa mRNA complexes as determined from three independent experiments. 5′-end-labeled coa mRNA (0.1 nM) was incubated with unlabeled RNAIII (20 nM), RNAIII-Δ7-9 (20 nM), and RNAIII-Δ13 (20 nM) at 37°C. Aliquots were withdrawn at various times (from 0 to 350 sec). The percentage of free coa mRNA was plotted as a function of time to estimate the association rate constant according to [40]. The values for the binding rate constants are the means of three independent experiments: 1.1×105 M−1 s−1 (RNAIII), 9.5×105 M−1 s−1 (RNAIII-Δ7-9), and 1.1×104 M−1 s−1 (RNAIII-Δ13).

Mentions: The contribution of the two binding sites toward complex formation was further evaluated by gel shift assays. Each experiment has been reproduced four times. In vitro labeled coa mRNA was first incubated with increasing concentrations of RNAIII or its variants (RNAIII-Δ13, RNAIII-Δ7–9, and the 3′ domain) at 37°C for 15 min (Fig. 4A). This experiment shows that coa mRNA binds to RNAIII or its 3′ domain with a Kd value of around 10 nM. The deletion of hairpins 7 to 9 in RNAIII had only a two-fold effect on the dissociation constant (around 25 nM), while the deletion of hairpin 13 in RNAIII increased significantly the Kd value by one order of magnitude (around 150 nM).


Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

Chevalier C, Boisset S, Romilly C, Masquida B, Fechter P, Geissmann T, Vandenesch F, Romby P - PLoS Pathog. (2010)

RNAIII binds efficiently to coa mRNA in vitro.(A) Determination of the apparent dissociation constant for RNAIII-coa mRNA complex. 5′-end-labeled coa mRNA was incubated alone (−) or with various concentrations of unlabeled wild type RNAIII, the 3′ domain, RNAIII-Δ7-9, and RNAIII-Δ13 (1, 5, 10, 20, 50, 100, 200, and 250 nM). The fraction of labeled coa mRNA associated with RNAIII or its derivatives was calculated from the counts in the corresponding band relative to the total counts in the lane. The Kd value was estimated as the concentration of RNAIII allowing 50% of coa mRNA binding. (B) Binding rate constant for various RNAIII-coa mRNA complexes as determined from three independent experiments. 5′-end-labeled coa mRNA (0.1 nM) was incubated with unlabeled RNAIII (20 nM), RNAIII-Δ7-9 (20 nM), and RNAIII-Δ13 (20 nM) at 37°C. Aliquots were withdrawn at various times (from 0 to 350 sec). The percentage of free coa mRNA was plotted as a function of time to estimate the association rate constant according to [40]. The values for the binding rate constants are the means of three independent experiments: 1.1×105 M−1 s−1 (RNAIII), 9.5×105 M−1 s−1 (RNAIII-Δ7-9), and 1.1×104 M−1 s−1 (RNAIII-Δ13).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837412&req=5

ppat-1000809-g004: RNAIII binds efficiently to coa mRNA in vitro.(A) Determination of the apparent dissociation constant for RNAIII-coa mRNA complex. 5′-end-labeled coa mRNA was incubated alone (−) or with various concentrations of unlabeled wild type RNAIII, the 3′ domain, RNAIII-Δ7-9, and RNAIII-Δ13 (1, 5, 10, 20, 50, 100, 200, and 250 nM). The fraction of labeled coa mRNA associated with RNAIII or its derivatives was calculated from the counts in the corresponding band relative to the total counts in the lane. The Kd value was estimated as the concentration of RNAIII allowing 50% of coa mRNA binding. (B) Binding rate constant for various RNAIII-coa mRNA complexes as determined from three independent experiments. 5′-end-labeled coa mRNA (0.1 nM) was incubated with unlabeled RNAIII (20 nM), RNAIII-Δ7-9 (20 nM), and RNAIII-Δ13 (20 nM) at 37°C. Aliquots were withdrawn at various times (from 0 to 350 sec). The percentage of free coa mRNA was plotted as a function of time to estimate the association rate constant according to [40]. The values for the binding rate constants are the means of three independent experiments: 1.1×105 M−1 s−1 (RNAIII), 9.5×105 M−1 s−1 (RNAIII-Δ7-9), and 1.1×104 M−1 s−1 (RNAIII-Δ13).
Mentions: The contribution of the two binding sites toward complex formation was further evaluated by gel shift assays. Each experiment has been reproduced four times. In vitro labeled coa mRNA was first incubated with increasing concentrations of RNAIII or its variants (RNAIII-Δ13, RNAIII-Δ7–9, and the 3′ domain) at 37°C for 15 min (Fig. 4A). This experiment shows that coa mRNA binds to RNAIII or its 3′ domain with a Kd value of around 10 nM. The deletion of hairpins 7 to 9 in RNAIII had only a two-fold effect on the dissociation constant (around 25 nM), while the deletion of hairpin 13 in RNAIII increased significantly the Kd value by one order of magnitude (around 150 nM).

Bottom Line: 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.It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, Strasbourg, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus