Small RNAs and Hfq capture unfolded RNA target sites during transcription.

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli.

Traffic of molecules across the bacterial membrane mainly relies on porins and transporters, whose expression must adapt to environmental conditions. To ensure bacterial fitness, synthesis and assembly of functional porins and transporters are regulated through a plethora of mechanisms. Among them, small regulatory RNAs (sRNAs) are known to be powerful post-transcriptional regulators. In Escherichia coli, the MicF sRNA is known to regulate only four targets, a very narrow targetome for a sRNA responding to various stresses, such as membrane stress, osmotic shock, or thermal shock. Using an in vivo pull-down assay combined with high-throughput RNA sequencing, we sought to identify new targets of MicF to better understand its role in the maintenance of cellular homoeostasis. Here, we report the first positively regulated target of MicF, the oppA mRNA. The OppA protein is the periplasmic component of the Opp ATP-binding cassette (ABC) oligopeptide transporter and regulates the import of short peptides, some of them bactericides. Mechanistic studies suggest that oppA translation is activated by MicF through a mechanism of action involving facilitated access to a translation-enhancing region in oppA 5'UTR. Intriguingly, MicF activation of oppA translation depends on cross-regulation by negative trans-acting effectors, the GcvB sRNA and the RNA chaperone protein Hfq.

Translational regulation by Hfq-Crc assemblies emerges from polymorphic ribonucleoprotein folding.

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo-EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.

Multifaceted Interplay between Hfq and the Small RNA GssA in Pseudomonas aeruginosa.

Behind the pathogenic lifestyle of Pseudomonas aeruginosa exists a complex regulatory network of intertwined switches at both the transcriptional and posttranscriptional levels. Major players that mediate translation regulation of several genes involved in host-P. aeruginosa interaction are small RNAs (sRNAs) and the Hfq protein. The canonical role of Hfq in sRNA-driven regulation is to act as a matchmaker between sRNAs and target mRNAs. Besides, the sRNA CrcZ is known to sequester Hfq and abrogate its function of translation repression of target mRNAs. In this study, we describe the novel sRNA GssA in the strain PA14 and its multifaceted interplay with Hfq. We show that GssA is multiresponsive to environmental and physiological signals and acts as an apical repressor of key bacterial functions in the human host such as the production of pyocyanin, utilization of glucose, and secretion of exotoxin A. We suggest that the main role of Hfq is not to directly assist GssA in its regulatory role but to repress GssA expression. In the case of pyocyanin production, we suggest that Hfq interplays with GssA also by converging a positive effect on this pathway. Furthermore, our results indicate that both Hfq and GssA play a positive role in anaerobic growth, possibly by regulating the respiratory chain. On the other hand, we show that GssA can modulate not only Hfq expression at both transcriptional and posttranscriptional levels but also that of CrcZ, thus potentially influencing the pleiotropic role of Hfq. IMPORTANCE The pathogenic lifestyle of the bacterium Pseudomonas aeruginosa, a leading cause of life-threatening infections in the airways of cystic fibrosis patients, is based on the fine regulation of virulence-associated factors. Regulatory small RNAs (sRNAs) and the RNA-binding protein Hfq are recognized key components within the P. aeruginosa regulatory networks involved in host-pathogen interaction. In this study, we characterized in the highly virulent P. aeruginosa strain PA14 the novel sRNA GssA. We found that it can establish a many-sided reciprocal interplay with Hfq which goes beyond the canonical mechanism of direct physical interaction that had previously been characterized for other sRNAs. Given that the Hfq-driven regulatory network of virulence factors is very broad and important for the progression of infection, we consider GssA as a new RNA target that can potentially be used to develop new antibacterial drugs.

Heterotypic phase separation of Hfq is linked to its roles as an RNA chaperone.

Hfq, an Sm-like protein and the major RNA chaperone in E. coli, has been shown to distribute non-uniformly along a helical path under normal growth conditions and to relocate to the cell poles under certain stress conditions. We have previously shown that Hfq relocation to the poles is accompanied by polar accumulation of most small RNAs (sRNAs). Here, we show that Hfq undergoes RNA-dependent phase separation to form cytoplasmic or polar condensates of different density under normal and stress conditions, respectively. Purified Hfq forms droplets in the presence of crowding agents or RNA, indicating that its condensation is via heterotypic interactions. Stress-induced relocation of Hfq condensates and sRNAs to the poles depends on the pole-localizer TmaR. Phase separation of Hfq correlates with its ability to perform its posttranscriptional roles as sRNA-stabilizer and sRNA-mRNA matchmaker. Our study offers a spatiotemporal mechanism for sRNA-mediated regulation in response to environmental changes.

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

Expanding the genetic toolkit helps dissect a global stress response in the early-branching species Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress-response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress-response network governed by the extracytoplasmic function sigma factor, σE. To this aim, we developed several genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a σE response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones, such as Hfq, we uncovered conservation of the noncoding arm of the σE response in form of the noncoding RNA FoxI. This regulatory small RNA acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σE. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium.

Synthetic Genetic Interactions Reveal a Dense and Cryptic Regulatory Network of Small Noncoding RNAs in Escherichia coli.

Over the past 20 years, we have learned that bacterial small noncoding RNAs (sRNAs) can rapidly effect changes in gene expression in response to stress. However, the broader role and impact of sRNA-mediated regulation in promoting bacterial survival has remained elusive. Indeed, there are few examples where disruption of sRNA-mediated gene regulation results in a discernible change in bacterial growth or survival. The lack of phenotypes attributable to loss of sRNA function suggests that either sRNAs are wholly dispensable or functional redundancies mask the impact of deleting a single sRNA. We investigated synthetic genetic interactions among sRNA genes in Escherichia coli by constructing pairwise deletions in 54 genes, including 52 sRNAs. Some 1,373 double deletion strains were studied for growth defects under 32 different nutrient stress conditions and revealed 1,131 genetic interactions. In one example, we identified a profound synthetic lethal interaction between ArcZ and CsrC when E. coli was grown on pyruvate, lactate, oxaloacetate, or d-/l-alanine, and we provide evidence that the expression of ppsA is dysregulated in the double deletion background, causing the conditionally lethal phenotype. This work employs a unique platform for studying sRNA-mediated gene regulation and sheds new light on the genetic network of sRNAs that underpins bacterial growth. IMPORTANCE sRNAs have long been purported to be a critical mechanism by which bacteria respond to stress; however, uncovering growth phenotypes for sRNA deletion strains in E. coli and related bacteria has proven particularly challenging. In contrast, the deletion of hfq, a chaperone required for the activity of many sRNAs in E. coli, results in striking growth defects in E. coli under a variety of medium conditions and chemical stressors. Here, we examined the importance of hfq and sRNA deletion strains for E. coli growth in nutrient-limited medium supplemented with 30 different carbon sources. We then systematically combined sRNA deletion mutations, creating a library of 1,373 sRNA double deletion strains, which we screened for growth under the same conditions, yielding 43,936 individual growth measurements. Our data uncovered more than 1,000 growth phenotypes for sRNA double deletion strains, shedding light on complicated networks of sRNA regulation that underpin bacterial survival under nutrient stress.

Dynamic Refolding of OxyS sRNA by the Hfq RNA Chaperone.

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5' and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.

Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.

Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions.

RNA-guided regulation of gene expression is found in all cell types. In this mode of regulation, antisense interactions between the regulatory RNA and its target are typically facilitated by a protein partner. Single-molecule fluorescence microscopy is a powerful tool for dissecting the conformational states and intermediates that contribute to target recognition. This chapter describes protocols for studying target recognition by bacterial small RNAs and their chaperone Hfq on the single-molecule level, using a total internal reflection fluorescence microscope. The sections cover the design of suitable RNA substrates for sRNA-mRNA annealing reactions, preparation of internally labeled mRNA for detecting conformational changes in the target, and key steps of the data analysis. These protocols can be adapted to other RNA-binding proteins that chaperone RNA interactions.

Diversity of bacterial small RNAs drives competitive strategies for a mutual chaperone.

Hundreds of bacterial small RNAs (sRNAs) require the Hfq chaperone to regulate mRNA expression. Hfq is limiting, thus competition among sRNAs for binding to Hfq shapes the proteomes of individual cells. To understand how sRNAs compete for a common partner, we present a single-molecule fluorescence platform to simultaneously visualize binding and release of multiple sRNAs with Hfq. We show that RNA residents rarely dissociate on their own. Instead, clashes between residents and challengers on the same face of Hfq cause rapid exchange, whereas RNAs that recognize different surfaces may cohabit Hfq for several minutes before one RNA departs. The prevalence of these pathways depends on the structure of each RNA and how it interacts with Hfq. We propose that sRNA diversity creates many pairwise interactions with Hfq that allow for distinct biological outcomes: active exchange favors fast regulation whereas co-residence of dissimilar RNAs favors target co-recognition or target exclusion.

Bacterial RNA chaperones and chaperone-like riboregulators: behind the scenes of RNA-mediated regulation of cellular metabolism.

In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins - Hfq, ProQ, and CsrA - have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism.

An overview of gene regulation in bacteria by small RNAs derived from mRNA 3' ends.

Over the past two decades, small noncoding RNAs (sRNAs) that regulate mRNAs by short base pairing have gone from a curiosity to a major class of post-transcriptional regulators in bacteria. They are integral to many stress responses and regulatory circuits, affecting almost all aspects of bacterial life. Following pioneering sRNA searches in the early 2000s, the field quickly focused on conserved sRNA genes in the intergenic regions of bacterial chromosomes. Yet, it soon emerged that there might be another rich source of bacterial sRNAs-processed 3' end fragments of mRNAs. Several such 3' end-derived sRNAs have now been characterized, often revealing unexpected, conserved functions in diverse cellular processes. Here, we review our current knowledge of these 3' end-derived sRNAs-their biogenesis through ribonucleases, their molecular mechanisms, their interactions with RNA-binding proteins such as Hfq or ProQ and their functional scope, which ranges from acting as specialized regulators of single metabolic genes to constituting entire noncoding arms in global stress responses. Recent global RNA interactome studies suggest that the importance of functional 3' end-derived sRNAs has been vastly underestimated and that this type of cross-regulation between genes at the mRNA level is more pervasive in bacteria than currently appreciated.

The core and accessory Hfq interactomes across Pseudomonas aeruginosa lineages.

The major RNA-binding protein Hfq interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria. However, studies tend to focus on single reference strains and assume that the findings may apply to the entire species, despite the important intra-species genetic diversity known to exist. Here, we use RIP-seq to identify Hfq-interacting RNAs in three strains representing the major phylogenetic lineages of Pseudomonas aeruginosa. We find that most interactions are in fact not conserved among the different strains. We identify growth phase-specific and strain-specific Hfq targets, including previously undescribed sRNAs. Strain-specific interactions are due to different accessory gene sets, RNA abundances, or potential context- or sequence- dependent regulatory mechanisms. The accessory Hfq interactome includes most mRNAs encoding Type III Secretion System (T3SS) components and secreted toxins in two strains, as well as a cluster of CRISPR guide RNAs in one strain. Conserved Hfq targets include the global virulence regulator Vfr and metabolic pathways involved in the transition from fast to slow growth. Furthermore, we use rGRIL-seq to show that RhlS, a quorum sensing sRNA, activates Vfr translation, thus revealing a link between quorum sensing and virulence regulation. Overall, our work highlights the important intra-species diversity in post-transcriptional regulatory networks in Pseudomonas aeruginosa.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Synthetic fused sRNA for the simultaneous repression of multiple genes.

Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.Key points• Synthetic fusion sRNA (fsRNA)-based system was constructed for the repression of multiple target genes.• fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.• The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).

Mobility of Bacterial Protein Hfq on dsDNA: Role of C-Terminus-Mediated Transient Binding.

The mobility of protein is fundamental in the machinery of life. Here, we have investigated the effect of DNA binding in conjunction with DNA segmental fluctuation (internal motion) of the bacterial Hfq master regulator devoid of its amyloid C-terminus domain. Hfq is one of the most abundant nucleoid associated proteins that shape the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy has been used to track a C-terminus domain lacking mutant form of Hfq on double-stranded DNA, which is stretched by confinement to a rectangular nanofluidic channel. The mobility of the mutant is strongly accelerated with respect to the wild-type variant. Furthermore, it shows a reverse dependence on the internal motion of DNA, in that slower motion results in slower protein diffusion. The results demonstrate the subtle role of DNA internal motion in controlling the mobility of a nucleoid associated protein, and, in particular, the importance of transient binding and moving DNA strands out of the way.

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq.

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

Global RNA interactome of Salmonella discovers a 5' UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity.

The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens.