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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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Enzymatic and chemical probing of the structure of the inhibitory RNAIII-coa mRNA complex.(A) Enzymatic hydrolysis of 5′-end-labeled 3′ domain of RNAIII, alone (lane 3) or in the presence of an excess of coa mRNA (lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to coa mRNA, respectively. Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. T1, V1: RNase T1 and RNase V1 hydrolysis, respectively. (B, C) Enzymatic hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or bound to the wild type RNAIII, or to the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9), of hairpin 13 (Δ13) or of hairpin 14 (Δ14). Concentrations of wild type or mutant RNAIII: lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM. Same legend as in A. (D) DEPC (N7A) modification of unlabeled coa mRNA, free (lane 3) or bound to the wild type RNAIII (lane 4), or the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9, lane 5), of hairpin 13 (Δ13, lane 6) or of hairpin 14 (Δ14, lane 7) at 200 nM. Lanes U, G, C, A: dideoxy-sequencing reactions performed on coa mRNA. (E) CMCT modification of unlabeled coa mRNA. Same legend as in D. Reactivity changes are indicated by bars on one side of each autoradiography. SD is for Shine-Dalgarno sequence and H7 is for hairpin 7 of RNAIII.
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ppat-1000809-g002: Enzymatic and chemical probing of the structure of the inhibitory RNAIII-coa mRNA complex.(A) Enzymatic hydrolysis of 5′-end-labeled 3′ domain of RNAIII, alone (lane 3) or in the presence of an excess of coa mRNA (lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to coa mRNA, respectively. Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. T1, V1: RNase T1 and RNase V1 hydrolysis, respectively. (B, C) Enzymatic hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or bound to the wild type RNAIII, or to the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9), of hairpin 13 (Δ13) or of hairpin 14 (Δ14). Concentrations of wild type or mutant RNAIII: lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM. Same legend as in A. (D) DEPC (N7A) modification of unlabeled coa mRNA, free (lane 3) or bound to the wild type RNAIII (lane 4), or the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9, lane 5), of hairpin 13 (Δ13, lane 6) or of hairpin 14 (Δ14, lane 7) at 200 nM. Lanes U, G, C, A: dideoxy-sequencing reactions performed on coa mRNA. (E) CMCT modification of unlabeled coa mRNA. Same legend as in D. Reactivity changes are indicated by bars on one side of each autoradiography. SD is for Shine-Dalgarno sequence and H7 is for hairpin 7 of RNAIII.

Mentions: The predicted base-pairing between RNAIII and coa mRNA and the in vivo experiments suggested that the RNAIII-dependent repression of coa mRNA was governed by direct RNAIII-mRNA pairing. We thus mapped the regions of interactions using enzymatic and chemical probing. The conformation of coa mRNA was probed using RNase T1 (specific for unpaired guanines), RNase V1 (specific for helical regions), and several base-specific chemicals such as dimethylsufate (methylates N1A≫N3C), a carbodiimide derivative (modifies N3U≫N1G), and diethylpyrocarbonate (carboxyethylates N7A). Several experiments of RNA structure probing are shown in Fig. 2. The secondary structure model of coa mRNA, which explains most of the probing data, is comprised of three stem-loop structures connected by unpaired residues (Fig. 3A). The AU-rich hairpin I is of weak stability but is proposed to occur based on the enzymatic cleavage pattern. However, the coexistence of alternative structures in the region encompassing nucleotides 10 to 70 may explain the concomitant presence of RNase V1 cleavages and the reactivity of many nucleotides at one of their Watson-Crick positions. In contrast, the long hairpin structure III located in the coding region of coa mRNA is well supported by the enzymatic cleavage patterns and the non reactivity of the Watson-Crick position of A77 to U85 towards chemicals (Fig. 2B-E). Binding of RNAIII induced changes in the region encompassing the ribosome binding site (RBS, nucleotides U10 to A48). RNAIII protected the guanines of the Shine-Dalgarno (SD) sequence against RNase T1 as well as the nucleotides U10 to U18 and A40 to A48 against chemical modifications (Fig. 2D, E). Concomitantly, RNAIII binding induced new RNase V1 cleavages at positions 39–41 and enhanced reactivity of A21, A24, A25, A29 to A31 at position N1, of A21 at position N7, and of U26 and U27 at position N3 in coa mRNA (Fig. 2D–E, Fig. 3B). These reactivity changes in the RBS of coa mRNA most likely resulted from the binding of the hairpin 13 of RNAIII because its deletion in RNAIII conferred no additional effect on the accessibility of the RBS of coa mRNA (Fig. 2C). Binding of coa mRNA to the 3′ domain or to RNAIII induced correlated changes in hairpin 13. Strong protections were observed at G441 against RNase T1 and at positions 411–415 and 448–449 against RNase V1 (Fig. 2A). Concomitantly, increased RNase V1 cleavages were observed at positions 433–434 and 444. All these data are thus consistent with the formation of a RNAIII-mRNA duplex that sequestered the RBS of coa mRNA (Fig. 3). This imperfect duplex involves two consecutive regions of 13 base-pairings interrupted by an internal loop and a bulged adenine 21 (Fig. 3B).


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)

Enzymatic and chemical probing of the structure of the inhibitory RNAIII-coa mRNA complex.(A) Enzymatic hydrolysis of 5′-end-labeled 3′ domain of RNAIII, alone (lane 3) or in the presence of an excess of coa mRNA (lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to coa mRNA, respectively. Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. T1, V1: RNase T1 and RNase V1 hydrolysis, respectively. (B, C) Enzymatic hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or bound to the wild type RNAIII, or to the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9), of hairpin 13 (Δ13) or of hairpin 14 (Δ14). Concentrations of wild type or mutant RNAIII: lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM. Same legend as in A. (D) DEPC (N7A) modification of unlabeled coa mRNA, free (lane 3) or bound to the wild type RNAIII (lane 4), or the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9, lane 5), of hairpin 13 (Δ13, lane 6) or of hairpin 14 (Δ14, lane 7) at 200 nM. Lanes U, G, C, A: dideoxy-sequencing reactions performed on coa mRNA. (E) CMCT modification of unlabeled coa mRNA. Same legend as in D. Reactivity changes are indicated by bars on one side of each autoradiography. SD is for Shine-Dalgarno sequence and H7 is for hairpin 7 of RNAIII.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837412&req=5

ppat-1000809-g002: Enzymatic and chemical probing of the structure of the inhibitory RNAIII-coa mRNA complex.(A) Enzymatic hydrolysis of 5′-end-labeled 3′ domain of RNAIII, alone (lane 3) or in the presence of an excess of coa mRNA (lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to coa mRNA, respectively. Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. T1, V1: RNase T1 and RNase V1 hydrolysis, respectively. (B, C) Enzymatic hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or bound to the wild type RNAIII, or to the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9), of hairpin 13 (Δ13) or of hairpin 14 (Δ14). Concentrations of wild type or mutant RNAIII: lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM. Same legend as in A. (D) DEPC (N7A) modification of unlabeled coa mRNA, free (lane 3) or bound to the wild type RNAIII (lane 4), or the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9, lane 5), of hairpin 13 (Δ13, lane 6) or of hairpin 14 (Δ14, lane 7) at 200 nM. Lanes U, G, C, A: dideoxy-sequencing reactions performed on coa mRNA. (E) CMCT modification of unlabeled coa mRNA. Same legend as in D. Reactivity changes are indicated by bars on one side of each autoradiography. SD is for Shine-Dalgarno sequence and H7 is for hairpin 7 of RNAIII.
Mentions: The predicted base-pairing between RNAIII and coa mRNA and the in vivo experiments suggested that the RNAIII-dependent repression of coa mRNA was governed by direct RNAIII-mRNA pairing. We thus mapped the regions of interactions using enzymatic and chemical probing. The conformation of coa mRNA was probed using RNase T1 (specific for unpaired guanines), RNase V1 (specific for helical regions), and several base-specific chemicals such as dimethylsufate (methylates N1A≫N3C), a carbodiimide derivative (modifies N3U≫N1G), and diethylpyrocarbonate (carboxyethylates N7A). Several experiments of RNA structure probing are shown in Fig. 2. The secondary structure model of coa mRNA, which explains most of the probing data, is comprised of three stem-loop structures connected by unpaired residues (Fig. 3A). The AU-rich hairpin I is of weak stability but is proposed to occur based on the enzymatic cleavage pattern. However, the coexistence of alternative structures in the region encompassing nucleotides 10 to 70 may explain the concomitant presence of RNase V1 cleavages and the reactivity of many nucleotides at one of their Watson-Crick positions. In contrast, the long hairpin structure III located in the coding region of coa mRNA is well supported by the enzymatic cleavage patterns and the non reactivity of the Watson-Crick position of A77 to U85 towards chemicals (Fig. 2B-E). Binding of RNAIII induced changes in the region encompassing the ribosome binding site (RBS, nucleotides U10 to A48). RNAIII protected the guanines of the Shine-Dalgarno (SD) sequence against RNase T1 as well as the nucleotides U10 to U18 and A40 to A48 against chemical modifications (Fig. 2D, E). Concomitantly, RNAIII binding induced new RNase V1 cleavages at positions 39–41 and enhanced reactivity of A21, A24, A25, A29 to A31 at position N1, of A21 at position N7, and of U26 and U27 at position N3 in coa mRNA (Fig. 2D–E, Fig. 3B). These reactivity changes in the RBS of coa mRNA most likely resulted from the binding of the hairpin 13 of RNAIII because its deletion in RNAIII conferred no additional effect on the accessibility of the RBS of coa mRNA (Fig. 2C). Binding of coa mRNA to the 3′ domain or to RNAIII induced correlated changes in hairpin 13. Strong protections were observed at G441 against RNase T1 and at positions 411–415 and 448–449 against RNase V1 (Fig. 2A). Concomitantly, increased RNase V1 cleavages were observed at positions 433–434 and 444. All these data are thus consistent with the formation of a RNAIII-mRNA duplex that sequestered the RBS of coa mRNA (Fig. 3). This imperfect duplex involves two consecutive regions of 13 base-pairings interrupted by an internal loop and a bulged adenine 21 (Fig. 3B).

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