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A-to-I mRNA editing controls disulfide bond formation and the toxicity of the HokB toxin in bacteria

ABSTRACT: Protein disulfide bonds between cysteine residues serve a prominent role in bacterial protein function, virulence, and viability. Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information at the RNA level. Previously, we discovered that A-to-I mRNA editing occurs in bacteria (Escherichia coli), identified the mediating enzyme, and showed it may introduce cysteine codons at the mRNA of multiple proteins. However, the ability of A-to-I mRNA editing to recode protein sequence, control disulfide bond formation, and affect its function was never investigated in bacteria. Here, we show that A-to-I mRNA editing constitutes a novel mechanism to control disulfide bond formation that can affect bacterial viability and growth. We focused on the most edited mRNA in E. coli that encodes the self, inner membrane toxin HokB in the HokB/sokB toxin-antitoxin system. First, we showed that A-to-I mRNA editing increases the toxicity of HokB, by recoding a tyrosine to a cysteine at the protein level. Next, we demonstrated that other (DNA-coded) cysteines in HokB and the ability of E. coli to form disulfide bonds are essential to the toxicity of edited HokB. We further showed that edited HokB can induce bacterial death and early entrance to stationary phase. Finally, structural modeling suggests that the mRNA editing-dependent cysteine of one HokB monomer interacts with a DNA-coded cysteine of another monomer, possibly stabilizing the HokB pore. Our work opens new avenues of research in how mRNA editing affects disulfide bond formation in different proteins and their role in bacterial biology.

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ORGANISM(S): Escherichia Coli

SUBMITTER: Dganit Melamed Kadosh

LAB HEAD: Dan Bar Yaacov

PROVIDER: PXD051162 | Pride | 2025-07-14

REPOSITORIES: Pride

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A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function.

Didi Liron L Fargeon Ofir O Aspit Liam L Elias Eyal E Braverman Dor D Melamed Dganit D Keidar-Friedman Daniel D Sorek Nadav N Raz Orit O Tamir Sharon Ovnat SO Zarivach Raz R Sal-Man Neta N Dahan Orna O Pilpel Yitzhak Y Bar Yaacov Dan D

Nucleic acids research 20250601 12

Adenosine-to-inosine (A-to-I) mRNA editing can alter genetic information at the RNA level and is known to affect protein sequence and function in eukaryotes. However, the ability of A-to-I mRNA editing to recode protein sequences in bacteria was never shown at the protein level. Furthermore, the effect of A-to-I mRNA editing itself-not by A-to-G DNA-mimicking mutations-on protein function was never demonstrated in bacteria. Here, we show at the RNA and protein levels that A-to-I mRNA editing dir ...[more]

PMID: 40598894

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Project description:Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3â?² untranslated region (3â?² UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stemâ??loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3â?² UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-Î³ and tumor-necrosis-factor-Î± induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases. 1) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in poly(A) RNA-seq data derived from endothelial cell transcriptome after ADAR1 or ADAR2 knockdown (n=2 biological replicates per condition, total n=8 biological samples). 2) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from peripheral blood mononuclear cells (n=12 total biological samples; n=4 replicates per condition). 3) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions (n=2 biological replicates per condition, total n=4 biological samples). 4) Evaluation of RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions after ADAR1 knockdown (n=3 replicates per condition, total n=12 biological samples). 5) HuR iCLIP RNA-sequencing data derived from HUVEC HuR iCLIP after ADAR1 knockdown (scrambled control and siADAR1, n=1 per condition, total n=2 biological samples).

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	Seq84934_88min_480Ex1.msf	Msf
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