The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage.
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.
Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.Show MeSH
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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).
Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.