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Link to original content: https://pubmed.ncbi.nlm.nih.gov/15678100/
Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression - PubMed Skip to main page content
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. 2005 Feb 23;24(4):824-35.
doi: 10.1038/sj.emboj.7600572. Epub 2005 Jan 27.

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Eric Huntzinger  1 , Sandrine Boisset, Cosmin Saveanu, Yvonne Benito, Thomas Geissmann, Abdelkader Namane, Gérard Lina, Jerome Etienne, Bernard Ehresmann, Chantal Ehresmann, Alain Jacquier, François Vandenesch, Pascale Romby
Affiliations

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Eric Huntzinger et al. EMBO J. .

Abstract

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell-wall-associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII-mediated inhibition of translation and degradation of the stable spa mRNA by the double-strand-specific endoribonuclease III (RNase III). The 3' end domain of RNAIII, partially complementary to the 5' part of spa mRNA, efficiently anneals to spa mRNA through an initial loop-loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

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Figures

Figure 1
Figure 1
β-galactosidase activity detected from different gene fusions. (A) Pspa∷lacZ and Pspa (+1/+12)∷lacZ fusions in S. aureus RN6390 (agr+, WT), WA400 (ΔrnaIII) and RN6390-Δrnc (LUG774, Δrnc), and PrpoB∷lacZ fusion in S. aureus RN6390 and WA400. (B) PrpoB∷spa (+1/+63)-lacZ fusion in different S. aureus strains: RN6390, WA400, WA400+RNAIII (LUG581, ΔrnaIII/pLUG298), WA400+RNAIII-Δ1 (LUG451, ΔrnaIII/pLUG302) and LUG774. The β-galactosidase activity is expressed in arbitrary unit per milligram of protein. The results represented a mean of three independent experiments.
Figure 2
Figure 2
RNAIII binds efficiently to spa mRNA in vitro. (A) The secondary structure model of RNAIII from Benito et al (2000), and the 3′ domain are given. The two mutants, which carry deletion in loop 13 (RNAIII-Δ1), and the whole deletion of hairpin 13 (RNAIII-Δ2) are squared. Nucleotides complementary to spa mRNA are printed in italic. (B) Determination of the apparent dissociation constant for RNAIII/spa mRNA complex. The 5′-end-labeled spa mRNA was incubated alone (−) or with various concentrations of unlabeled wild-type RNAIII (0.1, 0.5, 1, 5, 10, 50 and 100 nM) and mutant RNAIII (50 and 100 nM). The fraction of labeled spa mRNA associated with RNAIII was calculated from the counts in the corresponding band relative to the total counts in the lane. The apparent Kd value was determined as the concentration of RNAIII allowing 50% of spa mRNA binding. (C) Binding rate constant for the RNAIII/spa mRNA complex as determined from three independent experiments. The 5′-end-labeled RNAIII (0.1 nM) was incubated with unlabeled spa mRNA (1 nM) at 37°C. Aliquots were withdrawn at 0, 1, 2, 4, 8, 16, 32 and 60 min. The percentage of free RNAIII was plotted as a function of time.
Figure 3
Figure 3
RNAIII binds to the SD region of spa mRNA. (A, B) Enzymatic hydrolysis of 5′-end-labeled spa mRNA, free (−RNAIII) or in the presence of an excess of RNAIII (+RNAIII). (C) Chemical probing of unlabeled spa mRNA, free (−) or in complex with RNAIII (+). Lanes U, G, C and A: dideoxy-sequencing reactions performed on spa mRNA. (D) Enzymatic hydrolysis of 3′-end-labeled RNAIII free (−spa mRNA) or in the presence of an excess of spa mRNA (+spa mRNA). T2, T1 and V1: RNase T2, RNase T1 and RNase V1, respectively. Lanes T and L: RNase T1 under denaturing conditions and alkaline ladders, respectively. Lanes C: incubation controls in the absence of the probe. Arrows denote the main reactivity changes induced by complex formation.
Figure 4
Figure 4
Secondary structure models of free and complexed RNA. A summary of the probing results is represented on the secondary structure of spa mRNA (A) and RNAIII (B). Enzymatic cleavages are given as follows: RNase T1 (formula image), RNase T2 (formula image) and RNase V1 (formula image) moderate and (formula image) strong cleavage. Chemical modifications of cytosines at N3, and adenines at N1 towards DMS, and of uridines at N3 and guanines at N1 towards CMCT: full and dashed circled nucleotides are for strong and moderate reactivity, respectively. Small dots are for not determined due to unspecific cleavages or pauses of RT in the incubation control. Reactivity changes induced by complex formation are indicated as follows: black or empty circles denote strong and moderate protection, respectively; enhancements are represented by asterisks; new RNase V1 cleavages are shown by red arrows. Blue arrows show RNase III cleavages in the free RNA. Potential noncanonical base pairs are denoted by NoN. (C) Secondary structure model of the RNAIII/spa mRNA complex summarizing the enzymatic cleavages and chemical reactivities.
Figure 5
Figure 5
RNAIII prevents the formation of the ternary complex 30S-spa mRNA–tRNA. (A) Formation of the ternary complex between spa mRNA (15 nM), 30S ribosomal subunits (500 nM) and initiator tRNA (1 μM) was monitored in the absence (lane 5) or in the presence of wild-type RNAIII (lanes 6–8), RNAIII-Δ1 (lanes 9–11) and RNAIII-Δ2 (lanes 12–14). Concentrations of RNAIII species were 15, 75 and 150 nM. The toeprint at position +16 is indicated. Lanes U, G, C and A: dideoxy-sequencing reactions performed on spa mRNA. (B) Quantification of the data determined for three independent experiments. Relative toeprinting (toeprint band over full-length RNA+toeprint) was calculated by scanning of the gel with the Bio-imager Analyzer BAS 2000 (Fuji). The inhibition, given in %, represents the ratio between the relative toeprint in the presence of either the wild-type RNAIII or the mutant RNAs divided by the relative toeprint in the absence of RNAIII.
Figure 6
Figure 6
Analysis of spa mRNA and RNAIII levels in different strains. (A) Northern blot analysis on spa mRNA. RNAs from postexponential phase cultures were hybridized with probes corresponding to RNAIII and spa mRNA. Lane 1, RN6390 (WT, agr+); lane 2, WA400 (ΔrnaIII); lane 3, WA400+3′ domain (LUG484: ΔrnaIII/pLUG324); lane 4, WA400+RNAIII (LUG581: ΔrnaIII/pLUG298); lane 5, WA400+RNAIII-Δ1 (LUG451: ΔrnaIII/pLUG302); lane 6, RN6390-Δrnc (LUG774: Δrnc). (B) Measurements of the half-life of spa mRNA in the presence of rifampicin (300 μg/ml) in the strains WA400, RN6390-Δrnc and RN6390. For comparison, the half-life of spa-lacZ mRNA (agr-independent PrpoB promoter) in WA400 is given. (C) Measurements of the half-life of RNAIII in the presence of rifampicin in the strains RN6390 (WT) and RN6390-Δrnc. The half-life of 5S rRNA was measured on all the membranes in parallel, as an internal control. The quantity of spa mRNA or of RNAIII was normalized using the quantity of 5S rRNA at each time. The half-life was given as the time where 50% of the RNA was degraded. The experiments were reproduced four times.
Figure 7
Figure 7
S. aureus RNase III recognizes the free spa mRNA and RNAIII, and RNAIII–spa mRNA complex. (A) RNase III hydrolysis of 5′-end-labeled spa mRNA, free (−) or in the presence of an excess of RNAIII (+). (B) RNase III hydrolysis of 5′-end-labeled RNAIII, free (−) or in the presence of an excess of spa mRNA (+). Lanes C: incubation control in the absence of RNase III. Lanes T1, L: RNase T1 and alkaline ladders, respectively. Arrows pointed the RNase cleavages occurring either in the free RNA (formula image) or as the result of spa–RNAIII complex formation (formula image). (C) S. aureus RNase III binds specifically to RNAIII. Crude extract was loaded onto the free streptavidin beads (−) and on the beads bound to either the 3′ end biotinylated RNAIII (+RNAIII), or a group II intron fragment RNA (Δ52XBA). Elution was carried out with increasing concentrations of NaCl. M is for molecular weight markers (in kDa).
Figure 8
Figure 8
S. aureus RNAIII is a target of Hfq protein. (A) RNAIII co-immunoprecipitates with Hfq in vivo. RNAIII was detected by RT–PCR reaction in the flow through fraction (lane F) and in the precipitate fraction (lane E). The immunoprecipitation assays were also carried out with unspecific Ab, which do not bind Hfq. (B) Gel mobility shift assay with end-labeled spa mRNA in the presence of increasing concentrations of Hfq, and (C) with end-labeled RNAIII either free or in complex with spa mRNA in the presence of increasing concentrations of Hfq protein. Labeled RNAIII was first incubated with spa mRNA (10 nM) at 37°C for 20 min and various concentrations of Hfq (0.1, 5 and 1 μM) were added for a further 10 min of incubation. Note that the shift is not pronounced due to the large size of the RNAIII, but was reproducibly found in three independent experiments.
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