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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 acnB 3′ end is essential for RyhB-induced mRNA degradation. (A) Northern blot of total RNA of transcriptional acnB2688-lacZ fusion hybridized with acnB RNA probe. RNA was extracted after 10 min of RyhB induction with arabinose (0.05% final) or by Dip (200 μM final) (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). RyhB sRNA was hybridized to compare its expression in both conditions. 16S rRNA was used as loading control. (B) β-Galactosidase assays were performed with translational AcnB2688-LacZ fusion, which excluded acnB 3′UTR and its last two codons. Dip (200 μM final) for WT strain (EM1055) or arabinose (0.05% final) for pBAD-ryhB transformed strain (EM1455) were added to induce RyhB expression at an OD600 of 0.1. Specific β-galactosidase activity was measured in triplicates. Mean and SD of relative β-galactosidase activity (normalized to conditions without RyhB) are shown on graph. The empty vector pNM12 (EM1455) or ΔryhB (EM1238) strains were used as a control for AcnB2688-LacZ without RyhB. Northern blot with RyhB probe on total RNA was performed at an OD600 of 0.5 along with β-galactosidase assays. 5S rRNA was used as loading control.
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Figure 4: The acnB 3′ end is essential for RyhB-induced mRNA degradation. (A) Northern blot of total RNA of transcriptional acnB2688-lacZ fusion hybridized with acnB RNA probe. RNA was extracted after 10 min of RyhB induction with arabinose (0.05% final) or by Dip (200 μM final) (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). RyhB sRNA was hybridized to compare its expression in both conditions. 16S rRNA was used as loading control. (B) β-Galactosidase assays were performed with translational AcnB2688-LacZ fusion, which excluded acnB 3′UTR and its last two codons. Dip (200 μM final) for WT strain (EM1055) or arabinose (0.05% final) for pBAD-ryhB transformed strain (EM1455) were added to induce RyhB expression at an OD600 of 0.1. Specific β-galactosidase activity was measured in triplicates. Mean and SD of relative β-galactosidase activity (normalized to conditions without RyhB) are shown on graph. The empty vector pNM12 (EM1455) or ΔryhB (EM1238) strains were used as a control for AcnB2688-LacZ without RyhB. Northern blot with RyhB probe on total RNA was performed at an OD600 of 0.5 along with β-galactosidase assays. 5S rRNA was used as loading control.

Mentions: The data described above suggested that binding of AcnB to acnB 3′UTR interfered with RyhB-induced degradation of the mRNA. To add support to this interpretation, we constructed a transcriptional fusion (acnB2688-lacZ) comprising all but the last two codons of acnB ORF (Figure 3A). The reasoning behind the use of this construct was that it would allow RyhB binding to the translation initiation region (Supplementary Figures S2A, S2B and S2C) but would prevent apo-AcnB binding at the 3′UTR of acnB mRNA. The fusion acnB2688-lacZ was tested in cells grown under usual conditions (Figure 1A). Northern blots showed that acnB2688-lacZ remained resistant to the expression of RyhB after 10 min of induction (Figure 4A, lanes 1 and 2).


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

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

The acnB 3′ end is essential for RyhB-induced mRNA degradation. (A) Northern blot of total RNA of transcriptional acnB2688-lacZ fusion hybridized with acnB RNA probe. RNA was extracted after 10 min of RyhB induction with arabinose (0.05% final) or by Dip (200 μM final) (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). RyhB sRNA was hybridized to compare its expression in both conditions. 16S rRNA was used as loading control. (B) β-Galactosidase assays were performed with translational AcnB2688-LacZ fusion, which excluded acnB 3′UTR and its last two codons. Dip (200 μM final) for WT strain (EM1055) or arabinose (0.05% final) for pBAD-ryhB transformed strain (EM1455) were added to induce RyhB expression at an OD600 of 0.1. Specific β-galactosidase activity was measured in triplicates. Mean and SD of relative β-galactosidase activity (normalized to conditions without RyhB) are shown on graph. The empty vector pNM12 (EM1455) or ΔryhB (EM1238) strains were used as a control for AcnB2688-LacZ without RyhB. Northern blot with RyhB probe on total RNA was performed at an OD600 of 0.5 along with β-galactosidase assays. 5S rRNA was used as loading control.
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Figure 4: The acnB 3′ end is essential for RyhB-induced mRNA degradation. (A) Northern blot of total RNA of transcriptional acnB2688-lacZ fusion hybridized with acnB RNA probe. RNA was extracted after 10 min of RyhB induction with arabinose (0.05% final) or by Dip (200 μM final) (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). RyhB sRNA was hybridized to compare its expression in both conditions. 16S rRNA was used as loading control. (B) β-Galactosidase assays were performed with translational AcnB2688-LacZ fusion, which excluded acnB 3′UTR and its last two codons. Dip (200 μM final) for WT strain (EM1055) or arabinose (0.05% final) for pBAD-ryhB transformed strain (EM1455) were added to induce RyhB expression at an OD600 of 0.1. Specific β-galactosidase activity was measured in triplicates. Mean and SD of relative β-galactosidase activity (normalized to conditions without RyhB) are shown on graph. The empty vector pNM12 (EM1455) or ΔryhB (EM1238) strains were used as a control for AcnB2688-LacZ without RyhB. Northern blot with RyhB probe on total RNA was performed at an OD600 of 0.5 along with β-galactosidase assays. 5S rRNA was used as loading control.
Mentions: The data described above suggested that binding of AcnB to acnB 3′UTR interfered with RyhB-induced degradation of the mRNA. To add support to this interpretation, we constructed a transcriptional fusion (acnB2688-lacZ) comprising all but the last two codons of acnB ORF (Figure 3A). The reasoning behind the use of this construct was that it would allow RyhB binding to the translation initiation region (Supplementary Figures S2A, S2B and S2C) but would prevent apo-AcnB binding at the 3′UTR of acnB mRNA. The fusion acnB2688-lacZ was tested in cells grown under usual conditions (Figure 1A). Northern blots showed that acnB2688-lacZ remained resistant to the expression of RyhB after 10 min of induction (Figure 4A, lanes 1 and 2).

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