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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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AcnB3xFLAG binds to acnB RNA 3′UTR in vitro and in vivo in Fe-depleted medium. (A) Secondary structure and sequence of the 3′UTR of acnB mRNA. In red, nt protected by apo-AcnB in footprint experiment (see panel B). (B) Footprint analysis of purified AcnB3xFLAG protein showing binding site on acnB 3′UTR RNA. (Lane 1) NaOH ladder. (Lane 2) RNase T1 ladder. (Lane 3) RNase TA ladder. (Lanes 4–7) acnB RNA T1 digestion with addition of increasing amounts of purified AcnB3xFLAG protein (ratio acnB RNA:AcnB3xFLAG, 1:0, 1:1, 1:2 and 1:10, respectively). (Lanes 8–11) TA digestion of acnB RNA with addition of increasing amounts of AcnB3x FLAG protein (same ratio as used for T1 digestion). (C) AcnB3xFLAG binds to acnB mRNA in Fe-depleted medium in vivo. RNA IP performed with pBAD-acnB3xFLAG (JAB146A) (lanes 1, 2, 4 and 5) compared to pBAD-acnB (JAB151) (lanes 3 and 6). Dip (200 μM) was added 10 min before stopping cells growth and proceeding to RNA IP. Northern blots, hybridized with an acnB RNA probe and western blot, using an anti-AcnB antibody, were performed on samples before (input) and after (output) RNA IP. 16S rRNA and hns mRNA were used as negative controls for AcnB3xFLAG enrichment. Northern blot signals were quantified by densitometry and normalized to acnB mRNA levels without Dip. Mean and SD values from five biological replicates are shown. Statistical one-way ANOVA test significance is shown by asterisks (P < 0.002).
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Figure 2: AcnB3xFLAG binds to acnB RNA 3′UTR in vitro and in vivo in Fe-depleted medium. (A) Secondary structure and sequence of the 3′UTR of acnB mRNA. In red, nt protected by apo-AcnB in footprint experiment (see panel B). (B) Footprint analysis of purified AcnB3xFLAG protein showing binding site on acnB 3′UTR RNA. (Lane 1) NaOH ladder. (Lane 2) RNase T1 ladder. (Lane 3) RNase TA ladder. (Lanes 4–7) acnB RNA T1 digestion with addition of increasing amounts of purified AcnB3xFLAG protein (ratio acnB RNA:AcnB3xFLAG, 1:0, 1:1, 1:2 and 1:10, respectively). (Lanes 8–11) TA digestion of acnB RNA with addition of increasing amounts of AcnB3x FLAG protein (same ratio as used for T1 digestion). (C) AcnB3xFLAG binds to acnB mRNA in Fe-depleted medium in vivo. RNA IP performed with pBAD-acnB3xFLAG (JAB146A) (lanes 1, 2, 4 and 5) compared to pBAD-acnB (JAB151) (lanes 3 and 6). Dip (200 μM) was added 10 min before stopping cells growth and proceeding to RNA IP. Northern blots, hybridized with an acnB RNA probe and western blot, using an anti-AcnB antibody, were performed on samples before (input) and after (output) RNA IP. 16S rRNA and hns mRNA were used as negative controls for AcnB3xFLAG enrichment. Northern blot signals were quantified by densitometry and normalized to acnB mRNA levels without Dip. Mean and SD values from five biological replicates are shown. Statistical one-way ANOVA test significance is shown by asterisks (P < 0.002).

Mentions: Previous in vitro experiments have suggested that apo-AcnB bound to the 3′UTR of acnB mRNA (5). In these experiments, the authors used a 308 nucleotide-long sequence covering the 3′UTR of acnB without giving experimental evidence for the 3′ end of the transcript. We set up experiments to define the 3′ end of acnB by performing 3′RACE experiments, which indicated that the transcript terminated 67 nt after acnB ORF (Figure 2A, sequence and structure of acnB 3′UTR). Next, we performed experiments to identify the apo-AcnB binding site on acnB 3′UTR. Footprinting assays in which radiolabeled acnB 3′UTR was subjected to partial cleavage by RNase T1 (cleaves single-stranded Gs) and RNase TA (cleaves single-stranded As) in the presence of increasing amounts of purified AcnB3xFLAG protein were performed. We found clear protection of the structure surrounding the stem–loop located in 3′UTR (Figure 2B, lanes 4–11). These results suggested that apo-AcnB bound to the 3′UTR of acnB mRNA in the stem–loop structure immediately downstream of the ORF (red nucleotides in Figure 2A).


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

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

AcnB3xFLAG binds to acnB RNA 3′UTR in vitro and in vivo in Fe-depleted medium. (A) Secondary structure and sequence of the 3′UTR of acnB mRNA. In red, nt protected by apo-AcnB in footprint experiment (see panel B). (B) Footprint analysis of purified AcnB3xFLAG protein showing binding site on acnB 3′UTR RNA. (Lane 1) NaOH ladder. (Lane 2) RNase T1 ladder. (Lane 3) RNase TA ladder. (Lanes 4–7) acnB RNA T1 digestion with addition of increasing amounts of purified AcnB3xFLAG protein (ratio acnB RNA:AcnB3xFLAG, 1:0, 1:1, 1:2 and 1:10, respectively). (Lanes 8–11) TA digestion of acnB RNA with addition of increasing amounts of AcnB3x FLAG protein (same ratio as used for T1 digestion). (C) AcnB3xFLAG binds to acnB mRNA in Fe-depleted medium in vivo. RNA IP performed with pBAD-acnB3xFLAG (JAB146A) (lanes 1, 2, 4 and 5) compared to pBAD-acnB (JAB151) (lanes 3 and 6). Dip (200 μM) was added 10 min before stopping cells growth and proceeding to RNA IP. Northern blots, hybridized with an acnB RNA probe and western blot, using an anti-AcnB antibody, were performed on samples before (input) and after (output) RNA IP. 16S rRNA and hns mRNA were used as negative controls for AcnB3xFLAG enrichment. Northern blot signals were quantified by densitometry and normalized to acnB mRNA levels without Dip. Mean and SD values from five biological replicates are shown. Statistical one-way ANOVA test significance is shown by asterisks (P < 0.002).
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Figure 2: AcnB3xFLAG binds to acnB RNA 3′UTR in vitro and in vivo in Fe-depleted medium. (A) Secondary structure and sequence of the 3′UTR of acnB mRNA. In red, nt protected by apo-AcnB in footprint experiment (see panel B). (B) Footprint analysis of purified AcnB3xFLAG protein showing binding site on acnB 3′UTR RNA. (Lane 1) NaOH ladder. (Lane 2) RNase T1 ladder. (Lane 3) RNase TA ladder. (Lanes 4–7) acnB RNA T1 digestion with addition of increasing amounts of purified AcnB3xFLAG protein (ratio acnB RNA:AcnB3xFLAG, 1:0, 1:1, 1:2 and 1:10, respectively). (Lanes 8–11) TA digestion of acnB RNA with addition of increasing amounts of AcnB3x FLAG protein (same ratio as used for T1 digestion). (C) AcnB3xFLAG binds to acnB mRNA in Fe-depleted medium in vivo. RNA IP performed with pBAD-acnB3xFLAG (JAB146A) (lanes 1, 2, 4 and 5) compared to pBAD-acnB (JAB151) (lanes 3 and 6). Dip (200 μM) was added 10 min before stopping cells growth and proceeding to RNA IP. Northern blots, hybridized with an acnB RNA probe and western blot, using an anti-AcnB antibody, were performed on samples before (input) and after (output) RNA IP. 16S rRNA and hns mRNA were used as negative controls for AcnB3xFLAG enrichment. Northern blot signals were quantified by densitometry and normalized to acnB mRNA levels without Dip. Mean and SD values from five biological replicates are shown. Statistical one-way ANOVA test significance is shown by asterisks (P < 0.002).
Mentions: Previous in vitro experiments have suggested that apo-AcnB bound to the 3′UTR of acnB mRNA (5). In these experiments, the authors used a 308 nucleotide-long sequence covering the 3′UTR of acnB without giving experimental evidence for the 3′ end of the transcript. We set up experiments to define the 3′ end of acnB by performing 3′RACE experiments, which indicated that the transcript terminated 67 nt after acnB ORF (Figure 2A, sequence and structure of acnB 3′UTR). Next, we performed experiments to identify the apo-AcnB binding site on acnB 3′UTR. Footprinting assays in which radiolabeled acnB 3′UTR was subjected to partial cleavage by RNase T1 (cleaves single-stranded Gs) and RNase TA (cleaves single-stranded As) in the presence of increasing amounts of purified AcnB3xFLAG protein were performed. We found clear protection of the structure surrounding the stem–loop located in 3′UTR (Figure 2B, lanes 4–11). These results suggested that apo-AcnB bound to the 3′UTR of acnB mRNA in the stem–loop structure immediately downstream of the ORF (red nucleotides in Figure 2A).

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