Bacterial RNase III: Targets and physiology.

Bacteria can rapidly adapt to changes in their environment thanks to the innate flexibility of their genetic expression. The high turnover rate of RNAs, in particular messenger and regulatory RNAs, provides an important contribution to this dynamic adjustment. Recycling of RNAs is ensured by ribonucleases, among which RNase III is the focus of this review. RNase III enzymes are highly conserved from prokaryotes to eukaryotes and have the specific ability to cleave double-stranded RNAs. The role of RNase III in bacterial physiology has remained poorly explored for a long time. However, transcriptomic approaches recently uncovered a large impact of RNase III in gene expression in a wide range of bacteria, generating renewed interest in the physiological role of RNase III. In this review, we first describe the RNase III targets identified from global approaches in 8 bacterial species within 4 Phyla. We then present the conserved and unique functions of bacterial RNase III focusing on growth, resistance to stress, biofilm formation, motility and virulence. Altogether, this review highlights the underestimated impact of RNase III in bacterial adaptation.

Regulatory Interplay between RNase III and Antisense RNAs in E. coli: the Case of AsflhD and FlhD, Component of the Master Regulator of Motility.

In order to respond to ever-changing environmental cues, bacteria display resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases), and regulatory RNAs, including antisense RNAs (asRNAs). Bound to their complementary mRNA, asRNAs are primary targets for the double-strand-specific endoribonuclease, RNase III. Taking advantage of our own and previously published transcriptomic data sets obtained in strains inactivated for RNase III, we selected several candidate asRNAs and confirmed the existence of RNase III-sensitive asRNAs for crp, ompR, phoP, and flhD genes, all encoding global regulators of gene expression in Escherichia coli. Using FlhD, a component of the master regulator of motility (FlhD4C2), as our model, we demonstrate that the asRNA AsflhD, transcribed from the coding sequence of flhD, is involved in the fine-tuning of flhD expression and thus participates in the control of motility. IMPORTANCE The role of antisense RNAs (asRNAs) in the regulation of gene expression remains largely unexplored in bacteria. Here, we confirm that asRNAs can be part of layered regulatory networks, since some are found opposite to genes encoding global regulators. In particular, we show how an antisense RNA (AsflhD) to the flhD gene, encoding the transcription factor serving as the primary regulator of bacterial swimming motility (FlhD4C2), controls flhD expression, which in turn affects the expression of other genes of the motility cascade. The role of AsflhD highlights the importance of fine-tuning mechanisms mediated by asRNAs in the control of complex regulatory networks.

RNase III Participates in the Adaptation to Temperature Shock and Oxidative Stress in Escherichia coli.

Bacteria thrive in ever-changing environments by quickly remodeling their transcriptome and proteome via complex regulatory circuits. Regulation occurs at multiple steps, from the transcription of genes to the post-translational modification of proteins, via both protein and RNA regulators. At the post-transcriptional level, the RNA fate is balanced through the binding of ribosomes, chaperones and ribonucleases. We aim to decipher the role of the double-stranded-RNA-specific endoribonuclease RNase III and to evaluate its biological importance in the adaptation to modifications of the environment. The inactivation of RNase III affects a large number of genes and leads to several phenotypical defects, such as reduced thermotolerance in Escherichia coli. In this study, we reveal that RNase III inactivation leads to an increased sensitivity to temperature shock and oxidative stress. We further show that RNase III is important for the induction of the heat shock sigma factor RpoH and for the expression of the superoxide dismutase SodA.

Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli.

Bacterial small RNAs regulate the expression of multiple genes through imperfect base-pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram-negative bacteria. Recently, independent RNA-seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL-seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase-deficient E. coli strain (Δpeps) to Ala-Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively.

RNase III, Ribosome Biogenesis and Beyond.

The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood. Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles in ribosome biogenesis re-enforcing the importance of RNase III.

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

The world of asRNAs in Gram-negative and Gram-positive bacteria.

Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain essential functions and modulate accessory functions in response to environmental cues. Over the years, it has become clear that bacterial regulation of gene expression is still far from fully understood. This review focuses on antisense RNAs (asRNAs), a class of RNA regulators defined by their location in cis and their perfect complementarity with their targets, as opposed to small RNAs (sRNAs) which act in trans with only short regions of complementarity. For a long time, only few functional asRNAs in bacteria were known and were almost exclusively found on mobile genetic elements (MGEs), thus, their importance among the other regulators was underestimated. However, the extensive application of transcriptomic approaches has revealed the ubiquity of asRNAs in bacteria. This review aims to present the landscape of studied asRNAs in bacteria by comparing 67 characterized asRNAs from both Gram-positive and Gram-negative bacteria. First we describe the inherent ambiguity in the existence of asRNAs in bacteria, second, we highlight their diversity and their involvement in all aspects of bacterial life. Finally we compare their location and potential mode of action toward their target between Gram-negative and Gram-positive bacteria and present tendencies and exceptions that could lead to a better understanding of asRNA functions.

Physiological roles of antisense RNAs in prokaryotes.

Prokaryotes encounter constant and often brutal modifications to their environment. In order to survive, they need to maintain fitness, which includes adapting their protein expression patterns. Many factors control gene expression but this review focuses on just one, namely antisense RNAs (asRNAs), a class of non-coding RNAs (ncRNAs) characterized by their location in cis and their perfect complementarity with their targets. asRNAs were considered for a long time to be trivial and only to be found on mobile genetic elements. However, recent advances in methodology have revealed that their abundance and potential activities have been underestimated. This review aims to illustrate the role of asRNA in various physiologically crucial functions in both archaea and bacteria, which can be regrouped in three categories: cell maintenance, horizontal gene transfer and virulence. A literature survey of asRNAs demonstrates the difficulties to characterize and assign a role to asRNAs. With the aim of facilitating this task, we describe recent technological advances that could be of interest to identify new asRNAs and to discover their function.