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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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Apo-AcnB binding on acnB mRNA is essential for prevention of sRNA-induced degradation. (A) Scheme of acnB 3′UTR showing acnB2688, acnB2749, acnB2749-Mstem, acnB+20-3’UTR and acnBCC-stop-GCC transcriptional lacZ fusions constructs. The end of three commonly used transcriptional acnB’-lacZ fusion length 2688, 2749 and 2761 are annotated on the scheme. Letters in red corresponds to mutated nt for the indicated construct. (B) Northern blot of acnB2749 transcriptional lacZ fusion, sodB mRNA, RyhB sRNA and 16S rRNA that were hybridized with the corresponding RNA probes (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). Total RNA extraction was performed at the indicated time (0 or 10 min), at an OD600 of 0.5. Northern blots showing the effect of RyhB on acnB2749 (B), acnB2749-Mstem (C) and acnB2749-stem (D) transcriptional lacZ fusions. Expression of RyhB was induced at time 0 by addition of arabinose (0.05% final) or Dip (200 μM final). (E) AcnB3xFLAG RNA-IP on acnB2749, on acnB2749-Mstem and on acnB2749-stem constructs were expressed from pBAD33-derived plasmids in JAB292 cells (acnB736::kan). AcnB protein induction (OD600 of 0.1) was performed from pBAD-acnB3xFLAG and from pBAD24 plasmids. Then, when OD600 reached 0.5, RNA IP was performed. qRT-PCR analysis was performed with a probe covering the acnB 3′UTR for determination of acnB constructs enrichment. Mean with SD values from triplicates experiments are shown. Statistical one-way ANOVA test significance is shown by asterisks.
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Figure 3: Apo-AcnB binding on acnB mRNA is essential for prevention of sRNA-induced degradation. (A) Scheme of acnB 3′UTR showing acnB2688, acnB2749, acnB2749-Mstem, acnB+20-3’UTR and acnBCC-stop-GCC transcriptional lacZ fusions constructs. The end of three commonly used transcriptional acnB’-lacZ fusion length 2688, 2749 and 2761 are annotated on the scheme. Letters in red corresponds to mutated nt for the indicated construct. (B) Northern blot of acnB2749 transcriptional lacZ fusion, sodB mRNA, RyhB sRNA and 16S rRNA that were hybridized with the corresponding RNA probes (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). Total RNA extraction was performed at the indicated time (0 or 10 min), at an OD600 of 0.5. Northern blots showing the effect of RyhB on acnB2749 (B), acnB2749-Mstem (C) and acnB2749-stem (D) transcriptional lacZ fusions. Expression of RyhB was induced at time 0 by addition of arabinose (0.05% final) or Dip (200 μM final). (E) AcnB3xFLAG RNA-IP on acnB2749, on acnB2749-Mstem and on acnB2749-stem constructs were expressed from pBAD33-derived plasmids in JAB292 cells (acnB736::kan). AcnB protein induction (OD600 of 0.1) was performed from pBAD-acnB3xFLAG and from pBAD24 plasmids. Then, when OD600 reached 0.5, RNA IP was performed. qRT-PCR analysis was performed with a probe covering the acnB 3′UTR for determination of acnB constructs enrichment. Mean with SD values from triplicates experiments are shown. Statistical one-way ANOVA test significance is shown by asterisks.

Mentions: Results described above (Figure 2) suggested that acnB 3′UTR mediated the stability of acnB mRNA through interaction with AcnB under conditions of iron starvation. To investigate the involvement of acnB 3′UTR in AcnB binding, we engineered a transcriptional lacZ fusion, which carried all but the last 12 nt of acnB mRNA (fused at nt 2749, see Figure 3A). The fusion acnB2749-lacZ reproduced the same pattern of degradation previously observed (Figure 1A and B) 10 min after RyhB expression under control of the Ara-inducible promoter (Fe-rich condition) (Figure 3B, lanes 1 and 2) or in the presence of Dip (Figure 3B, lanes 5 and 6). Furthermore, data indicated (Figure 2B) that the stem–loop located immediately downstream of acnB 3′UTR could bind to AcnB under iron depletion. To investigate the role of this stem–loop in acnB 3′UTR, we constructed an acnB2749Mstem-lacZ fusion that was mutated for 2 nt within the stem structure. This mutation disrupts the stem–loop secondary structure as suggested by Mfold (http://mfold.rit.albany.edu/?q=mfold. Data not shown). Northern blots indicated that acnB2749Mstem-lacZ was completely degraded after 10 min of RyhB expression whether RyhB was expressed from plasmid with Ara (Figure 3C, lanes 1 and 2) or, endogenously, in the presence of Dip (Figure 3C, lanes 5 and 6). To address the question whether the RNA sequence or the stem–loop structure was responsible for the loss of regulation, we constructed a fusion carrying compensatory mutations that reconstituted the stem–loop in acnB 3′UTR (acnB2749-stem, Figure 3A). Northern blots revealed that, under typical growth conditions, the acnB2749-stem construct was degraded when RyhB was expressed from Ara (Figure 3D, lanes 1 and 2) but remained intact when RyhB was expressed in cells cultured in the presence of Dip (Figure 3D, lanes 5 and 6). These results were interpreted to suggest that the stem–loop structure located immediately downstream of acnB ORF played an important role in the stability of acnB mRNA when RyhB is expressed.


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

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

Apo-AcnB binding on acnB mRNA is essential for prevention of sRNA-induced degradation. (A) Scheme of acnB 3′UTR showing acnB2688, acnB2749, acnB2749-Mstem, acnB+20-3’UTR and acnBCC-stop-GCC transcriptional lacZ fusions constructs. The end of three commonly used transcriptional acnB’-lacZ fusion length 2688, 2749 and 2761 are annotated on the scheme. Letters in red corresponds to mutated nt for the indicated construct. (B) Northern blot of acnB2749 transcriptional lacZ fusion, sodB mRNA, RyhB sRNA and 16S rRNA that were hybridized with the corresponding RNA probes (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). Total RNA extraction was performed at the indicated time (0 or 10 min), at an OD600 of 0.5. Northern blots showing the effect of RyhB on acnB2749 (B), acnB2749-Mstem (C) and acnB2749-stem (D) transcriptional lacZ fusions. Expression of RyhB was induced at time 0 by addition of arabinose (0.05% final) or Dip (200 μM final). (E) AcnB3xFLAG RNA-IP on acnB2749, on acnB2749-Mstem and on acnB2749-stem constructs were expressed from pBAD33-derived plasmids in JAB292 cells (acnB736::kan). AcnB protein induction (OD600 of 0.1) was performed from pBAD-acnB3xFLAG and from pBAD24 plasmids. Then, when OD600 reached 0.5, RNA IP was performed. qRT-PCR analysis was performed with a probe covering the acnB 3′UTR for determination of acnB constructs enrichment. Mean with SD values from triplicates experiments are shown. Statistical one-way ANOVA test significance is shown by asterisks.
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Figure 3: Apo-AcnB binding on acnB mRNA is essential for prevention of sRNA-induced degradation. (A) Scheme of acnB 3′UTR showing acnB2688, acnB2749, acnB2749-Mstem, acnB+20-3’UTR and acnBCC-stop-GCC transcriptional lacZ fusions constructs. The end of three commonly used transcriptional acnB’-lacZ fusion length 2688, 2749 and 2761 are annotated on the scheme. Letters in red corresponds to mutated nt for the indicated construct. (B) Northern blot of acnB2749 transcriptional lacZ fusion, sodB mRNA, RyhB sRNA and 16S rRNA that were hybridized with the corresponding RNA probes (ARA: strain KP1135 carrying pGD3-ryhB (RyhB+) or empty pGD3 plasmid (RyhB−); Dip: strains EM1055 (WT) or EM1238 (ΔryhB)). Total RNA extraction was performed at the indicated time (0 or 10 min), at an OD600 of 0.5. Northern blots showing the effect of RyhB on acnB2749 (B), acnB2749-Mstem (C) and acnB2749-stem (D) transcriptional lacZ fusions. Expression of RyhB was induced at time 0 by addition of arabinose (0.05% final) or Dip (200 μM final). (E) AcnB3xFLAG RNA-IP on acnB2749, on acnB2749-Mstem and on acnB2749-stem constructs were expressed from pBAD33-derived plasmids in JAB292 cells (acnB736::kan). AcnB protein induction (OD600 of 0.1) was performed from pBAD-acnB3xFLAG and from pBAD24 plasmids. Then, when OD600 reached 0.5, RNA IP was performed. qRT-PCR analysis was performed with a probe covering the acnB 3′UTR for determination of acnB constructs enrichment. Mean with SD values from triplicates experiments are shown. Statistical one-way ANOVA test significance is shown by asterisks.
Mentions: Results described above (Figure 2) suggested that acnB 3′UTR mediated the stability of acnB mRNA through interaction with AcnB under conditions of iron starvation. To investigate the involvement of acnB 3′UTR in AcnB binding, we engineered a transcriptional lacZ fusion, which carried all but the last 12 nt of acnB mRNA (fused at nt 2749, see Figure 3A). The fusion acnB2749-lacZ reproduced the same pattern of degradation previously observed (Figure 1A and B) 10 min after RyhB expression under control of the Ara-inducible promoter (Fe-rich condition) (Figure 3B, lanes 1 and 2) or in the presence of Dip (Figure 3B, lanes 5 and 6). Furthermore, data indicated (Figure 2B) that the stem–loop located immediately downstream of acnB 3′UTR could bind to AcnB under iron depletion. To investigate the role of this stem–loop in acnB 3′UTR, we constructed an acnB2749Mstem-lacZ fusion that was mutated for 2 nt within the stem structure. This mutation disrupts the stem–loop secondary structure as suggested by Mfold (http://mfold.rit.albany.edu/?q=mfold. Data not shown). Northern blots indicated that acnB2749Mstem-lacZ was completely degraded after 10 min of RyhB expression whether RyhB was expressed from plasmid with Ara (Figure 3C, lanes 1 and 2) or, endogenously, in the presence of Dip (Figure 3C, lanes 5 and 6). To address the question whether the RNA sequence or the stem–loop structure was responsible for the loss of regulation, we constructed a fusion carrying compensatory mutations that reconstituted the stem–loop in acnB 3′UTR (acnB2749-stem, Figure 3A). Northern blots revealed that, under typical growth conditions, the acnB2749-stem construct was degraded when RyhB was expressed from Ara (Figure 3D, lanes 1 and 2) but remained intact when RyhB was expressed in cells cultured in the presence of Dip (Figure 3D, lanes 5 and 6). These results were interpreted to suggest that the stem–loop structure located immediately downstream of acnB ORF played an important role in the stability of acnB mRNA when RyhB is expressed.

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