Abstract
Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.
Highlights
RNA editing is a posttranscriptional event that alters genomic RNA sequences via insertion, deletion, deamination or substitution [1, 2]
Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes that has only been recently documented in bacteria
We show that tRNA-specific adenosine deaminase (TadA), which encodes adenosine deaminase, can directly bind to mRNA of target genes through recognition of a GACG motif
Summary
RNA editing is a posttranscriptional event that alters genomic RNA sequences via insertion, deletion, deamination or substitution [1, 2]. Adenosine-to-inosine (A-to-I) editing, which is catalyzed by the adenosine deaminase RNA-specific (ADAR) family of enzymes, is the most prevalent type of editing in metazoans [3, 4]. This form of editing functions in multiple biological processes ranging from nonsense-mediated mRNA decay and alternative splicing to gene expression and translation [5, 6]. Since inosine (I) is recognized as guanosine (G) by the polymerase enzyme and translational machinery, A-to-I editing in coding regions of the mRNA may lead to codon changes, resulting in further diversification of protein function [2]. In prokaryotes, A-to-I RNA editing of mRNA has rarely been reported
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