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.

The essential role of mRNA degradation in understanding and engineering E. coli metabolism.

Metabolic engineering strategies are crucial for the development of bacterial cell factories with improved performance. Until now, optimal metabolic networks have been designed based on systems biology approaches integrating large-scale data on the steady-state concentrations of mRNA, protein and metabolites, sometimes with dynamic data on fluxes, but rarely with any information on mRNA degradation. In this review, we compile growing evidence that mRNA degradation is a key regulatory level in E. coli that metabolic engineering strategies should take into account. We first discuss how mRNA degradation interacts with transcription and translation, two other gene expression processes, to balance transcription regulation and remove poorly translated mRNAs. The many reciprocal interactions between mRNA degradation and metabolism are also highlighted: metabolic activity can be controlled by changes in mRNA degradation and in return, the activity of the mRNA degradation machinery is controlled by metabolic factors. The mathematical models of the crosstalk between mRNA degradation dynamics and other cellular processes are presented and discussed with a view towards novel mRNA degradation-based metabolic engineering strategies. We show finally that mRNA degradation-based strategies have already successfully been applied to improve heterologous protein synthesis. Overall, this review underlines how important mRNA degradation is in regulating E. coli metabolism and identifies mRNA degradation as a key target for innovative metabolic engineering strategies in biotechnology.

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.

Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile.

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

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.

Identification of protein-protein and ribonucleoprotein complexes containing Hfq.

Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

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.

RNA polyadenylation and its consequences in prokaryotes.

Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin-antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile.

Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.

Landscape of RNA polyadenylation in E. coli.

Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5΄-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178 transcripts were either up- or down-regulated in the mutant when compared to the wild-type strain. Poly(A)polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A)polymerase was the accumulation of numerous sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications based on the sequence of unencoded 3΄-ends, was developed to identify polyadenylated molecules. Overall our results shed new light on the broad spectrum of action of polyadenylation on gene expression and demonstrate the importance of poly(A) dependent degradation to remove structured RNA fragments.

The small RNA SraG participates in PNPase homeostasis.

The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3'-5' exoribonuclease involved in mRNA decay in Escherichia coli The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.

A new custom microarray for sRNA profiling in Escherichia coli.

Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level.

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function.

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.

The interplay of Hfq, poly(A) polymerase I and exoribonucleases at the 3' ends of RNAs resulting from Rho-independent termination: A tentative model.

Discovered in eukaryotes as a modification essential for mRNA function, polyadenylation was then identified as a means used by all cells to destabilize RNA. In Escherichia coli, most accessible 3' RNA extremities are believed to be potential targets of poly(A) polymerase I. However, some RNAs might be preferentially adenylated. After a short statement of the current knowledge of poly(A) metabolism, we discuss how Hfq could affect recognition and polyadenylation of RNA terminated by Rho-independent terminators. Comparison of RNA terminus leads to the proposal that RNAs harboring 3' terminal features required for Hfq binding are not polyadenylated, whereas those lacking these structural elements can gain the oligo(A) tails that initiate exonucleolytic degradation. We also speculate that Hfq stimulates the synthesis of longer tails that could be used as Hfq-binding sites involved in non-characterized functions of Hfq-dependent sRNAs.

Role of polyadenylation in regulation of the flagella cascade and motility in Escherichia coli.

Polyadenylation is recognized as part of a surveillance machinery for eliminating defective RNA molecules in eukaryotes and prokaryotes. Escherichia coli strains, deficient in poly(A)polymerase I (PAP I), expressed less flagellin compared to wild-type strains. Because flagellin synthesis is a late step in the flagellar biosynthesis pathway, we assessed the role of PAP I in this cascade and in flagella function. Transcription of flhDC, fliA, and fliC was decreased in the PAP I mutant. These results provide evidence that polyadenylation positively controls the expression of genes belonging to the flagellar biosynthesis pathway and that this effect is mediated through the FlhDC master regulator. However, the downshift in flagella gene expression in the mutant strain did not provoke any noticeable defects in the synthesis of flagella, in biofilm formation and in swimming speed although there was a reduction in motility on soft agar. Our data support an alternative hypothesis that the reduced motility of the mutant resulted from an alteration of the cell membrane composition caused in part by the higher level of GlmS (Glucosamine-6P synthase) which accumulates in the mutant. In agreement with this hypothesis the mutant is more sensitive to hydrophobic agents and antibiotics and in particular to vancomycin. We propose that PAP I participates in the ability of the bacteria to adapt to and survive detrimental conditions by constantly monitoring and adjusting to its environment.

Multiple activities of RNA-binding proteins S1 and Hfq.

In all organisms, RNA-binding proteins participate in modulating all the steps in the life cycle of RNA, including transcription, folding, translation and turnover. In bacteria, RNA-binding proteins may be specific for a few RNA targets (e.g., several ribosomal proteins that recognize both rRNA during ribosome assembly and their own mRNAs when acting as highly specific autogenous repressors) or function as global regulators implicated in numerous regulatory networks. Some RNA-binding proteins combine all these features, and this particularly concerns the ribosomal protein S1 and the Sm-like protein Hfq. S1 is a key mRNA-binding protein in gram-negative bacteria; it recognizes mRNA leaders and provides binding of diverse mRNAs to the ribosome at the initiation step of translation. Moreover, S1 is a highly specific autogenous repressor that is able to distinguish its own mRNA from all the others. Hfq is recognized as a global regulator that facilitates small RNA-mRNA interactions in bacteria; it thereby controls the expression of many mRNAs either positively or negatively. In addition, these two proteins were reported to affect transcription, RNA degradation and other processes. Although they have no sequence specificity, Hfq and S1 preferentially bind A/U-rich single-stranded RNA regions; despite this, they nevertheless carry out very different tasks in the cell. This review is focused on the diversity of functions that can be performed by these abundant RNA-binding bacterial proteins.

Search for poly(A) polymerase targets in E. coli reveals its implication in surveillance of Glu tRNA processing and degradation of stable RNAs.

Polyadenylation is a universal post-transcriptional modification involved in degradation and quality control of bacterial RNAs. In Escherichia coli, it is admitted that any accessible RNA 3' end can be tagged by a poly(A) tail for decay. However, we do not have yet an overall view of the population of polyadenylated molecules. The sampling of polyadenylated RNAs presented here demonstrates that rRNA fragments and tRNA precursors originating from the internal spacer regions of the rrn operons, in particular, rrnB are abundant poly(A) polymerase targets. Focused analysis showed that Glu tRNA precursors originating from the rrnB and rrnG transcripts exhibit long 3' trailers that are primarily removed by PNPase and to a lesser extent by RNase II and poly(A) polymerase. Moreover, 3' trimming by exoribonucleases precedes 5' end maturation by RNase P. Interestingly, characterization of RNA fragments that accumulate in a PNPase deficient strain showed that Glu tRNA precursors still harbouring the 5' leader can be degraded by a 3' to 5' quality control pathway involving poly(A) polymerase. This demonstrates that the surveillance of tRNA maturation described for a defective tRNA also applies to a wild-type tRNA.