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The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage.

Benjamin JA, Massé E - Nucleic Acids Res. (2014)

Bottom Line: In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3'UTR and stabilize it when intracellular iron become scarce.Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site.This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.

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The mRNA acnB is post-transcriptionally regulated as a function of Fe status. (A) Northern blots analysis of RyhB and Fe starvation effects on acnB mRNA levels. Total RNA extracted from strains EM1455 (ΔryhB), carrying pBAD-ryhB, and EM1055 (WT) strains was hybridized with an acnB-specific probe. Cells were grown to an OD600 of 0.5 in LB medium at which point arabinose (0.05% final) (strain EM1455 carrying pBAD-ryhB, lanes 1–5), 2.2′-dipyridyl (Dip) (200 μM final) (strain EM1055, lanes 6–10) or both (strain 1455 carrying pBAD-ryhB, lanes 11–15) were added at time 0. Total RNA was extracted at the indicated time. The well-characterized RyhB target sodB mRNA is shown as a positive control for RyhB-induced degradation under all conditions. 16S rRNA was used as a loading control. (B) Same results as in panel (A) but total RNA extracted from strains EM1455 (ΔryhB) carrying pNM12 and EM1238 (ΔryhB) in which there was no RyhB expression. (C) Decay rates of acnB mRNA in LB medium. Fe chelator Dip (200 μM) was added at time −10 min. Then, rifampicin (Rif) was added (250 μg/ml final concentration) at time 0 before total RNAs were extracted at the indicated time points. These data are representative of four independent biological replicates quantified by densitometry and normalized to time 0 (0+) for both +Dip and −Dip. Mean and SD values experiments are shown. The dashed line corresponds to 50% decrease in mRNA levels. Inset: Northern blots using acnB probe showing acnB mRNA stability. Calculated acnB half-life value is indicated beside respective experimental conditions on the graph.
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Figure 1: The mRNA acnB is post-transcriptionally regulated as a function of Fe status. (A) Northern blots analysis of RyhB and Fe starvation effects on acnB mRNA levels. Total RNA extracted from strains EM1455 (ΔryhB), carrying pBAD-ryhB, and EM1055 (WT) strains was hybridized with an acnB-specific probe. Cells were grown to an OD600 of 0.5 in LB medium at which point arabinose (0.05% final) (strain EM1455 carrying pBAD-ryhB, lanes 1–5), 2.2′-dipyridyl (Dip) (200 μM final) (strain EM1055, lanes 6–10) or both (strain 1455 carrying pBAD-ryhB, lanes 11–15) were added at time 0. Total RNA was extracted at the indicated time. The well-characterized RyhB target sodB mRNA is shown as a positive control for RyhB-induced degradation under all conditions. 16S rRNA was used as a loading control. (B) Same results as in panel (A) but total RNA extracted from strains EM1455 (ΔryhB) carrying pNM12 and EM1238 (ΔryhB) in which there was no RyhB expression. (C) Decay rates of acnB mRNA in LB medium. Fe chelator Dip (200 μM) was added at time −10 min. Then, rifampicin (Rif) was added (250 μg/ml final concentration) at time 0 before total RNAs were extracted at the indicated time points. These data are representative of four independent biological replicates quantified by densitometry and normalized to time 0 (0+) for both +Dip and −Dip. Mean and SD values experiments are shown. The dashed line corresponds to 50% decrease in mRNA levels. Inset: Northern blots using acnB probe showing acnB mRNA stability. Calculated acnB half-life value is indicated beside respective experimental conditions on the graph.

Mentions: The acnB mRNA has been previously shown to be down-regulated by the sRNA RyhB when expressed from the arabinose-inducible promoter of the pBAD-ryhB construct (31). In these experiments, RyhB was induced independently of intracellular iron status and Fur activity, both of which normally control the expression of the endogenous ryhB promoter (38,39). Notably, AcnB has been shown to shift from catalytic (holo-AcnB) to RNA-binding regulatory mode (apo-AcnB) under conditions of iron starvation (4,5). Therefore, we asked whether acnB mRNA could be protected from RyhB-induced mRNA degradation when AcnB switched to binding its own mRNA (apo-form) under conditions of low Fe. We first addressed this question by inducing expression of RyhB under two different modes of induction. The first mode involved cultures in Fe-rich medium (Figure 1A, left panels) of EM1455 (ΔryhB) cells carrying an arabinose-inducible pBAD-ryhB plasmid (0.1% arabinose, Ara). The second mode involved EM1055 (WT) cells grown in Fe-poor medium by adding the iron chelator 2,2′-dipyridyl (Dip) and where RyhB is under the control of its endogenous promoter. Results showed that when RyhB was expressed in cells carrying the pBAD-ryhB plasmid in Fe-rich medium, it triggered a rapid degradation (within 5 min) of acnB mRNA (Figure 1A, lanes 1–5). The mRNA sodB was used as a positive control in these experiments and showed the expected degradation kinetics of a RyhB target (≤5 min). These results were in agreement with our previous observations that RyhB-induced degradation of acnB when expressed under the control of pBAD-ryhB when arabinose was present in the medium (31). In marked contrast, when RyhB sRNA was induced from its endogenous promoter under low Fe conditions, acnB mRNA remained stable even after 10 min of RyhB expression. This result was unexpected given that the fact that RyhB-sensitive sodB mRNA became fully degraded under similar conditions (Figure 1A, lanes 6–10). These results suggested that decreased intracellular iron levels may help to stabilize acnB mRNA although RyhB sRNA was fully expressed and able to induce degradation of control target sodB mRNA. To confirm that these results (Figure 1A, lanes 1–5) were not due to an arabinose-related effect, a third mode of induction was investigated. In this case, EM1455 cells carrying pBAD-ryhB were grown under condition of iron depletion (Dip, 250 μM) and were treated with arabinose to induce RyhB. Induction of RyhB under low iron showed that acnB mRNA remained stable throughout the experiment, although RyhB expression induced sodB degradation within 5 min (Figure 1A, lanes 11–15). These results confirmed that iron-depleted conditions protected acnB mRNA against RyhB-induced degradation.


The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage.

Benjamin JA, Massé E - Nucleic Acids Res. (2014)

The mRNA acnB is post-transcriptionally regulated as a function of Fe status. (A) Northern blots analysis of RyhB and Fe starvation effects on acnB mRNA levels. Total RNA extracted from strains EM1455 (ΔryhB), carrying pBAD-ryhB, and EM1055 (WT) strains was hybridized with an acnB-specific probe. Cells were grown to an OD600 of 0.5 in LB medium at which point arabinose (0.05% final) (strain EM1455 carrying pBAD-ryhB, lanes 1–5), 2.2′-dipyridyl (Dip) (200 μM final) (strain EM1055, lanes 6–10) or both (strain 1455 carrying pBAD-ryhB, lanes 11–15) were added at time 0. Total RNA was extracted at the indicated time. The well-characterized RyhB target sodB mRNA is shown as a positive control for RyhB-induced degradation under all conditions. 16S rRNA was used as a loading control. (B) Same results as in panel (A) but total RNA extracted from strains EM1455 (ΔryhB) carrying pNM12 and EM1238 (ΔryhB) in which there was no RyhB expression. (C) Decay rates of acnB mRNA in LB medium. Fe chelator Dip (200 μM) was added at time −10 min. Then, rifampicin (Rif) was added (250 μg/ml final concentration) at time 0 before total RNAs were extracted at the indicated time points. These data are representative of four independent biological replicates quantified by densitometry and normalized to time 0 (0+) for both +Dip and −Dip. Mean and SD values experiments are shown. The dashed line corresponds to 50% decrease in mRNA levels. Inset: Northern blots using acnB probe showing acnB mRNA stability. Calculated acnB half-life value is indicated beside respective experimental conditions on the graph.
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Figure 1: The mRNA acnB is post-transcriptionally regulated as a function of Fe status. (A) Northern blots analysis of RyhB and Fe starvation effects on acnB mRNA levels. Total RNA extracted from strains EM1455 (ΔryhB), carrying pBAD-ryhB, and EM1055 (WT) strains was hybridized with an acnB-specific probe. Cells were grown to an OD600 of 0.5 in LB medium at which point arabinose (0.05% final) (strain EM1455 carrying pBAD-ryhB, lanes 1–5), 2.2′-dipyridyl (Dip) (200 μM final) (strain EM1055, lanes 6–10) or both (strain 1455 carrying pBAD-ryhB, lanes 11–15) were added at time 0. Total RNA was extracted at the indicated time. The well-characterized RyhB target sodB mRNA is shown as a positive control for RyhB-induced degradation under all conditions. 16S rRNA was used as a loading control. (B) Same results as in panel (A) but total RNA extracted from strains EM1455 (ΔryhB) carrying pNM12 and EM1238 (ΔryhB) in which there was no RyhB expression. (C) Decay rates of acnB mRNA in LB medium. Fe chelator Dip (200 μM) was added at time −10 min. Then, rifampicin (Rif) was added (250 μg/ml final concentration) at time 0 before total RNAs were extracted at the indicated time points. These data are representative of four independent biological replicates quantified by densitometry and normalized to time 0 (0+) for both +Dip and −Dip. Mean and SD values experiments are shown. The dashed line corresponds to 50% decrease in mRNA levels. Inset: Northern blots using acnB probe showing acnB mRNA stability. Calculated acnB half-life value is indicated beside respective experimental conditions on the graph.
Mentions: The acnB mRNA has been previously shown to be down-regulated by the sRNA RyhB when expressed from the arabinose-inducible promoter of the pBAD-ryhB construct (31). In these experiments, RyhB was induced independently of intracellular iron status and Fur activity, both of which normally control the expression of the endogenous ryhB promoter (38,39). Notably, AcnB has been shown to shift from catalytic (holo-AcnB) to RNA-binding regulatory mode (apo-AcnB) under conditions of iron starvation (4,5). Therefore, we asked whether acnB mRNA could be protected from RyhB-induced mRNA degradation when AcnB switched to binding its own mRNA (apo-form) under conditions of low Fe. We first addressed this question by inducing expression of RyhB under two different modes of induction. The first mode involved cultures in Fe-rich medium (Figure 1A, left panels) of EM1455 (ΔryhB) cells carrying an arabinose-inducible pBAD-ryhB plasmid (0.1% arabinose, Ara). The second mode involved EM1055 (WT) cells grown in Fe-poor medium by adding the iron chelator 2,2′-dipyridyl (Dip) and where RyhB is under the control of its endogenous promoter. Results showed that when RyhB was expressed in cells carrying the pBAD-ryhB plasmid in Fe-rich medium, it triggered a rapid degradation (within 5 min) of acnB mRNA (Figure 1A, lanes 1–5). The mRNA sodB was used as a positive control in these experiments and showed the expected degradation kinetics of a RyhB target (≤5 min). These results were in agreement with our previous observations that RyhB-induced degradation of acnB when expressed under the control of pBAD-ryhB when arabinose was present in the medium (31). In marked contrast, when RyhB sRNA was induced from its endogenous promoter under low Fe conditions, acnB mRNA remained stable even after 10 min of RyhB expression. This result was unexpected given that the fact that RyhB-sensitive sodB mRNA became fully degraded under similar conditions (Figure 1A, lanes 6–10). These results suggested that decreased intracellular iron levels may help to stabilize acnB mRNA although RyhB sRNA was fully expressed and able to induce degradation of control target sodB mRNA. To confirm that these results (Figure 1A, lanes 1–5) were not due to an arabinose-related effect, a third mode of induction was investigated. In this case, EM1455 cells carrying pBAD-ryhB were grown under condition of iron depletion (Dip, 250 μM) and were treated with arabinose to induce RyhB. Induction of RyhB under low iron showed that acnB mRNA remained stable throughout the experiment, although RyhB expression induced sodB degradation within 5 min (Figure 1A, lanes 11–15). These results confirmed that iron-depleted conditions protected acnB mRNA against RyhB-induced degradation.

Bottom Line: In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3'UTR and stabilize it when intracellular iron become scarce.Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site.This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.

Show MeSH
Related in: MedlinePlus