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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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Both apo-AcnB stabilization and RyhB negative regulation affect AcnB levels. (A) Quantitative western blot of AcnB levels in Fe-depleted medium. Fe depletion was induced by addition of Dip (200 μM final) to WT (EM1055) or to ΔryhB strains (EM1238) at time 0. Total proteins were extracted at the indicated time (h). Relative densitometry levels are indicated under the lanes. (B) Quantitative western blot of AcnB levels under Fe-rich conditions when RyhB sRNA was expressed in WT strain (EM1455) from pBAD-ryhB compared with an empty plasmid pNM12 (0.01% arabinose final). Proteins were extracted at the indicated time (h). EF-Tu protein was used as a loading control in both experiments. A polyclonal anti-AcnB antibody was used for hybridization and antibody specificity was determined using a protein extract from ΔacnB strain (strain JAB284 for panel A, JAB154 for panel B). Relative densitometry levels are indicated under the lanes. A IRDye 800CW-conjugated secondary antibody was used for quantification.
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Figure 6: Both apo-AcnB stabilization and RyhB negative regulation affect AcnB levels. (A) Quantitative western blot of AcnB levels in Fe-depleted medium. Fe depletion was induced by addition of Dip (200 μM final) to WT (EM1055) or to ΔryhB strains (EM1238) at time 0. Total proteins were extracted at the indicated time (h). Relative densitometry levels are indicated under the lanes. (B) Quantitative western blot of AcnB levels under Fe-rich conditions when RyhB sRNA was expressed in WT strain (EM1455) from pBAD-ryhB compared with an empty plasmid pNM12 (0.01% arabinose final). Proteins were extracted at the indicated time (h). EF-Tu protein was used as a loading control in both experiments. A polyclonal anti-AcnB antibody was used for hybridization and antibody specificity was determined using a protein extract from ΔacnB strain (strain JAB284 for panel A, JAB154 for panel B). Relative densitometry levels are indicated under the lanes. A IRDye 800CW-conjugated secondary antibody was used for quantification.

Mentions: To evaluate the effect RyhB on AcnB expression, we monitored AcnB levels by western blots and compared the results to native AcnB from WT and ΔryhB cells grown under Fe starvation. Unexpectedly, AcnB levels remained stable over time when RyhB was induced in the presence of Dip (Figure 6A, left panel). In marked contrast, we observed a significant accumulation of AcnB (3.6-fold increase) in ΔryhB cells 2 h after Dip treatment (Figure 6A, right panel). These results suggested that RyhB expression significantly repressed AcnB translation when cells grew under low Fe conditions.


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

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

Both apo-AcnB stabilization and RyhB negative regulation affect AcnB levels. (A) Quantitative western blot of AcnB levels in Fe-depleted medium. Fe depletion was induced by addition of Dip (200 μM final) to WT (EM1055) or to ΔryhB strains (EM1238) at time 0. Total proteins were extracted at the indicated time (h). Relative densitometry levels are indicated under the lanes. (B) Quantitative western blot of AcnB levels under Fe-rich conditions when RyhB sRNA was expressed in WT strain (EM1455) from pBAD-ryhB compared with an empty plasmid pNM12 (0.01% arabinose final). Proteins were extracted at the indicated time (h). EF-Tu protein was used as a loading control in both experiments. A polyclonal anti-AcnB antibody was used for hybridization and antibody specificity was determined using a protein extract from ΔacnB strain (strain JAB284 for panel A, JAB154 for panel B). Relative densitometry levels are indicated under the lanes. A IRDye 800CW-conjugated secondary antibody was used for quantification.
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Figure 6: Both apo-AcnB stabilization and RyhB negative regulation affect AcnB levels. (A) Quantitative western blot of AcnB levels in Fe-depleted medium. Fe depletion was induced by addition of Dip (200 μM final) to WT (EM1055) or to ΔryhB strains (EM1238) at time 0. Total proteins were extracted at the indicated time (h). Relative densitometry levels are indicated under the lanes. (B) Quantitative western blot of AcnB levels under Fe-rich conditions when RyhB sRNA was expressed in WT strain (EM1455) from pBAD-ryhB compared with an empty plasmid pNM12 (0.01% arabinose final). Proteins were extracted at the indicated time (h). EF-Tu protein was used as a loading control in both experiments. A polyclonal anti-AcnB antibody was used for hybridization and antibody specificity was determined using a protein extract from ΔacnB strain (strain JAB284 for panel A, JAB154 for panel B). Relative densitometry levels are indicated under the lanes. A IRDye 800CW-conjugated secondary antibody was used for quantification.
Mentions: To evaluate the effect RyhB on AcnB expression, we monitored AcnB levels by western blots and compared the results to native AcnB from WT and ΔryhB cells grown under Fe starvation. Unexpectedly, AcnB levels remained stable over time when RyhB was induced in the presence of Dip (Figure 6A, left panel). In marked contrast, we observed a significant accumulation of AcnB (3.6-fold increase) in ΔryhB cells 2 h after Dip treatment (Figure 6A, right panel). These results suggested that RyhB expression significantly repressed AcnB translation when cells grew under low Fe conditions.

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