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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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RyhB induces RNase E cleavage in acnB mRNA 3′ end. (A) Northern blot of acnB mRNA were performed on total RNA extracted from strain EM1455 and JF133 (rne131 mutant). Both stains were transformed with plasmid allowing arabinose-dependent RyhB expression (pBAD-ryhB) or with control vector (pNM12). Cells were grown in LB medium to an OD600 of 0.5 and RyhB sRNA was induced at time 0 by addition of arabinose (0.01% final). Total RNA extraction was performed after 10 min of induction. (B) RNA degradosome degradation assay to map RNase E cleavage site on acnB 3′UTR. Effect of purified RNA degradosome (0.5 ng/μl final concentration) on 32P-pCp 3′ end labeling acnB-3′UTR (lanes 4 and 5) and acnB3′UTR-CC-stop-GCC (lanes 9 and 10). (Lanes 1 and 6) NaOH ladder. (Lanes 2 and 7) RNase T1 ladder. (Lanes 3 and 8) RNase TA ladder. (Lanes 4 and 9) Radiolabeled acnB RNA alone (see Figure 3A for cleavage site map). (C) qRT-PCR of acnBCC-stop-gcc transcriptional lacZ fusion from total RNA extraction. RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055). Total RNA extraction was performed at the indicated time, at mid-logarithmic growth phase. Endogenous sodB mRNA levels after RyhB expression are also shown. Mean and SD values of four replicates experiments are shown. Asterisks correspond to statistical significance from one-way ANOVA test. (D) Northern blot using acnB probe showing the effect of RyhB expression on acnB+20-3′UTR-transcriptional lacZ fusion (after 10 min). Before RNA extraction, RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055) at time 0. Empty plasmid pGD3 or ΔryhB strains were used as negatives controls for ARA and Dip panels, respectively.
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Figure 5: RyhB induces RNase E cleavage in acnB mRNA 3′ end. (A) Northern blot of acnB mRNA were performed on total RNA extracted from strain EM1455 and JF133 (rne131 mutant). Both stains were transformed with plasmid allowing arabinose-dependent RyhB expression (pBAD-ryhB) or with control vector (pNM12). Cells were grown in LB medium to an OD600 of 0.5 and RyhB sRNA was induced at time 0 by addition of arabinose (0.01% final). Total RNA extraction was performed after 10 min of induction. (B) RNA degradosome degradation assay to map RNase E cleavage site on acnB 3′UTR. Effect of purified RNA degradosome (0.5 ng/μl final concentration) on 32P-pCp 3′ end labeling acnB-3′UTR (lanes 4 and 5) and acnB3′UTR-CC-stop-GCC (lanes 9 and 10). (Lanes 1 and 6) NaOH ladder. (Lanes 2 and 7) RNase T1 ladder. (Lanes 3 and 8) RNase TA ladder. (Lanes 4 and 9) Radiolabeled acnB RNA alone (see Figure 3A for cleavage site map). (C) qRT-PCR of acnBCC-stop-gcc transcriptional lacZ fusion from total RNA extraction. RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055). Total RNA extraction was performed at the indicated time, at mid-logarithmic growth phase. Endogenous sodB mRNA levels after RyhB expression are also shown. Mean and SD values of four replicates experiments are shown. Asterisks correspond to statistical significance from one-way ANOVA test. (D) Northern blot using acnB probe showing the effect of RyhB expression on acnB+20-3′UTR-transcriptional lacZ fusion (after 10 min). Before RNA extraction, RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055) at time 0. Empty plasmid pGD3 or ΔryhB strains were used as negatives controls for ARA and Dip panels, respectively.

Mentions: Most trans-acting sRNAs that induce degradation of their target mRNAs usually recruit RNase E through Hfq-mediated interaction (40). To confirm this mechanism in relationship to RyhB and acnB, we performed pulse-expression of RyhB in rne131 background, which carried a deletion of RNase E C-terminus that prevented RNase E recruitment by Hfq. Northern blots confirmed that RNase E was essential for acnB mRNA degradation after a 10 min of RyhB induction with Ara (Figure 5A, lanes 2 and 4). The possibility that acnB 3′UTR carried a cleavage site recognized by RNase E (33) was investigated by degradation assays using purified RNA degradosome in the presence of radiolabeled acnB 3′UTR (from nt 2569 to nt 2767). Degradation assay revealed a strong cleavage signal near the stop codon of acnB ORF (Figure 5B, compare lanes 4 and 5), which mapped between nt 2690 and 2697 (see Figure 3A) close to acnB stop codon. We then mutagenized the cleavage site by modifying the sequence UUUAAAAA to CCUAAGCC (see Figure 3A). The mutant construct was then subjected to RNA degradosome assay. Results showed that CCUAAGCC mutations in the end of acnB ORF prevented cleavage by RNase E (Figure 5B, compare lanes 5 and 10). These results suggested that RNase E required the original AU-rich sequence near the stop codon of acnB mRNA to induce cleavage. To confirm that the RNase E-dependent cleavage site (Figure 5B) was essential, we mutated the acnB2749-lacZ construct to introduce the CCUAAGCC mutation that blocked RNase E cleavage. Cells harboring the construct acnB2749CC-stop-GCC were subjected to RyhB expression and data analyzed by qRT-PCR. The acnB2749CC-stop-GCC construct was completely resistant to RyhB-induced mRNA degradation under Fe-rich condition (Figure 5C, compare acnB2749CC-stop-GCC and sodB panels, columns 1 and 2), highlighting the importance of the RyhB-induced cleavage site that mapped at the end of acnB ORF (Figure 5B, nt 2692–2697).


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

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

RyhB induces RNase E cleavage in acnB mRNA 3′ end. (A) Northern blot of acnB mRNA were performed on total RNA extracted from strain EM1455 and JF133 (rne131 mutant). Both stains were transformed with plasmid allowing arabinose-dependent RyhB expression (pBAD-ryhB) or with control vector (pNM12). Cells were grown in LB medium to an OD600 of 0.5 and RyhB sRNA was induced at time 0 by addition of arabinose (0.01% final). Total RNA extraction was performed after 10 min of induction. (B) RNA degradosome degradation assay to map RNase E cleavage site on acnB 3′UTR. Effect of purified RNA degradosome (0.5 ng/μl final concentration) on 32P-pCp 3′ end labeling acnB-3′UTR (lanes 4 and 5) and acnB3′UTR-CC-stop-GCC (lanes 9 and 10). (Lanes 1 and 6) NaOH ladder. (Lanes 2 and 7) RNase T1 ladder. (Lanes 3 and 8) RNase TA ladder. (Lanes 4 and 9) Radiolabeled acnB RNA alone (see Figure 3A for cleavage site map). (C) qRT-PCR of acnBCC-stop-gcc transcriptional lacZ fusion from total RNA extraction. RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055). Total RNA extraction was performed at the indicated time, at mid-logarithmic growth phase. Endogenous sodB mRNA levels after RyhB expression are also shown. Mean and SD values of four replicates experiments are shown. Asterisks correspond to statistical significance from one-way ANOVA test. (D) Northern blot using acnB probe showing the effect of RyhB expression on acnB+20-3′UTR-transcriptional lacZ fusion (after 10 min). Before RNA extraction, RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055) at time 0. Empty plasmid pGD3 or ΔryhB strains were used as negatives controls for ARA and Dip panels, respectively.
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Figure 5: RyhB induces RNase E cleavage in acnB mRNA 3′ end. (A) Northern blot of acnB mRNA were performed on total RNA extracted from strain EM1455 and JF133 (rne131 mutant). Both stains were transformed with plasmid allowing arabinose-dependent RyhB expression (pBAD-ryhB) or with control vector (pNM12). Cells were grown in LB medium to an OD600 of 0.5 and RyhB sRNA was induced at time 0 by addition of arabinose (0.01% final). Total RNA extraction was performed after 10 min of induction. (B) RNA degradosome degradation assay to map RNase E cleavage site on acnB 3′UTR. Effect of purified RNA degradosome (0.5 ng/μl final concentration) on 32P-pCp 3′ end labeling acnB-3′UTR (lanes 4 and 5) and acnB3′UTR-CC-stop-GCC (lanes 9 and 10). (Lanes 1 and 6) NaOH ladder. (Lanes 2 and 7) RNase T1 ladder. (Lanes 3 and 8) RNase TA ladder. (Lanes 4 and 9) Radiolabeled acnB RNA alone (see Figure 3A for cleavage site map). (C) qRT-PCR of acnBCC-stop-gcc transcriptional lacZ fusion from total RNA extraction. RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055). Total RNA extraction was performed at the indicated time, at mid-logarithmic growth phase. Endogenous sodB mRNA levels after RyhB expression are also shown. Mean and SD values of four replicates experiments are shown. Asterisks correspond to statistical significance from one-way ANOVA test. (D) Northern blot using acnB probe showing the effect of RyhB expression on acnB+20-3′UTR-transcriptional lacZ fusion (after 10 min). Before RNA extraction, RyhB sRNA expression was induced by addition of arabinose (0.05% final) for pGD3-ryhB (KP1135) or by addition of Dip (200 μM final) for wild-type (EM1055) at time 0. Empty plasmid pGD3 or ΔryhB strains were used as negatives controls for ARA and Dip panels, respectively.
Mentions: Most trans-acting sRNAs that induce degradation of their target mRNAs usually recruit RNase E through Hfq-mediated interaction (40). To confirm this mechanism in relationship to RyhB and acnB, we performed pulse-expression of RyhB in rne131 background, which carried a deletion of RNase E C-terminus that prevented RNase E recruitment by Hfq. Northern blots confirmed that RNase E was essential for acnB mRNA degradation after a 10 min of RyhB induction with Ara (Figure 5A, lanes 2 and 4). The possibility that acnB 3′UTR carried a cleavage site recognized by RNase E (33) was investigated by degradation assays using purified RNA degradosome in the presence of radiolabeled acnB 3′UTR (from nt 2569 to nt 2767). Degradation assay revealed a strong cleavage signal near the stop codon of acnB ORF (Figure 5B, compare lanes 4 and 5), which mapped between nt 2690 and 2697 (see Figure 3A) close to acnB stop codon. We then mutagenized the cleavage site by modifying the sequence UUUAAAAA to CCUAAGCC (see Figure 3A). The mutant construct was then subjected to RNA degradosome assay. Results showed that CCUAAGCC mutations in the end of acnB ORF prevented cleavage by RNase E (Figure 5B, compare lanes 5 and 10). These results suggested that RNase E required the original AU-rich sequence near the stop codon of acnB mRNA to induce cleavage. To confirm that the RNase E-dependent cleavage site (Figure 5B) was essential, we mutated the acnB2749-lacZ construct to introduce the CCUAAGCC mutation that blocked RNase E cleavage. Cells harboring the construct acnB2749CC-stop-GCC were subjected to RyhB expression and data analyzed by qRT-PCR. The acnB2749CC-stop-GCC construct was completely resistant to RyhB-induced mRNA degradation under Fe-rich condition (Figure 5C, compare acnB2749CC-stop-GCC and sodB panels, columns 1 and 2), highlighting the importance of the RyhB-induced cleavage site that mapped at the end of acnB ORF (Figure 5B, nt 2692–2697).

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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Related in: MedlinePlus