Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation.

Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ may be detrimental.

Circulating tumor cell-derived exosome-transmitted long non-coding RNA TTN-AS1 can promote the proliferation and migration of cholangiocarcinoma cells.

BACKGROUND: Exosomes assume a pivotal role as essential mediators of intercellular communication within tumor microenvironments. Within this context, long noncoding RNAs (LncRNAs) have been observed to be preferentially sorted into exosomes, thus exerting regulatory control over the initiation and progression of cancer through diverse mechanisms. RESULTS: Exosomes were successfully isolated from cholangiocarcinoma (CCA) CTCs organoid and healthy human serum. Notably, the LncRNA titin-antisense RNA1 (TTN-AS1) exhibited a conspicuous up-regulation within CCA CTCs organoid derived exosomes. Furthermore, a significant elevation of TTN-AS1 expression was observed in tumor tissues, as well as in blood and serum exosomes from patients afflicted with CCA. Importantly, this hightened TTN-AS1 expression in serum exosomes of CCA patients manifested a strong correlation with both lymph node metastasis and TNM staging. Remarkably, both CCA CTCs organoid-derived exosomes and CCA cells-derived exosomes featuring pronounced TTN-AS1 expression demonstrated the capability to the proliferation and migratory potential of CCA cells. Validation of these outcomes was conducted in vivo experiments. CONCLUSIONS: In conclusion, our study elucidating that CCA CTCs-derived exosomes possess the capacity to bolster the metastasis tendencies of CCA cells by transporting TTN-AS1. These observations underscore the potential of TTN-AS1 within CTCs-derived exosomes to serve as a promising biomarker for the diagnosis and therapeutic management of CCA.

Small regulatory RNA RSaX28 promotes virulence by reinforcing the stability of RNAIII in community-associated ST398 clonotype Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a notorious pathogen that cause metastatic or complicated infections. Hypervirulent ST398 clonotype strains, remarkably increased in recent years, dominated Community-associated S. aureus (CA-SA) infections in the past decade in China. Small RNAs like RNAIII have been demonstrated to play important roles in regulating the virulence of S. aureus, however, the regulatory roles played by many of these sRNAs in the ST398 clonotype strains are still unclear. Through transcriptome screening and combined with knockout phenotype analysis, we have identified a highly transcribed sRNA, RSaX28, in the ST398 clonotype strains. Sequence analysis revealed that RSaX28 is highly conserved in the most epidemic clonotypes of S. aureus, but its high transcription level is particularly prominent in the ST398 clonotype strains. Characterization of RSaX28 through RACE and Northern blot revealed its length to be 533nt. RSaX28 is capable of promoting the hemolytic ability, reducing biofilm formation capacity, and enhancing virulence of S. aureus in the in vivo murine infection model. Through IntaRNA prediction and EMSA validation, we found that RSaX28 can specifically interact with RNAIII, promoting its stability and positively regulating the translation of downstream alpha-toxin while inhibiting the translation of Sbi, thereby regulating the virulence and biofilm formation capacity of the ST398 clonotype strains. RSaX28 is an important virulence regulatory factor in the ST398 clonotype S. aureus and represents a potential important target for future treatment and immune intervention against S. aureus infections.

A natural bacterial pathogen of C. elegans uses a small RNA to induce transgenerational inheritance of learned avoidance.

C. elegans can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans' natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans' natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in P. vranovensis, that has a 16-nucleotide match to an exon of the C. elegans gene maco-1. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in C. elegans' natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σE-dependent small RNA with unusual mRNA activation activity.

The oral commensal Fusobacterium nucleatum can spread to extra-oral sites, where it is associated with diverse pathologies, including pre-term birth and cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of the RNA regulatory networks that allow this bacterium to adapt to its various niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor, σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped the 5' and 3' ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156-nucleotide sRNA previously misannotated as the 5' untranslated region (UTR) of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as is FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5' and 3' UTRs in F. nucleatum and expands our understanding of the σE response in this evolutionarily distant bacterium.IMPORTANCEThe oral microbe Fusobacterium nucleatum can colonize secondary sites, including cancer tissue, and likely deploys complex regulatory systems to adapt to these new environments. These systems are largely unknown, partly due to the phylogenetic distance of F. nucleatum to other model organisms. Previously, we identified a global stress response mediated by σE that displays functional conservation with the envelope stress response in Proteobacteria, comprising a coding and noncoding regulatory arm. Through global identification of transcriptional start and stop sites, we uncovered the small RNA (sRNA) FoxJ as a novel component of the noncoding arm of the σE response in F. nucleatum. Together with its companion sRNA FoxI, FoxJ post-transcriptionally modulates the synthesis of envelope proteins, revealing a conserved function for σE-dependent sRNAs between Fusobacteriota and Proteobacteria. Moreover, FoxJ activates the gene expression for several targets, which is a mode of regulation previously unseen in the noncoding arm of the σE response.

In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria.

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described.

RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.

RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces.

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.

Bacterial small RNAs may mediate immune response differences seen in respiratory syncytial virus versus rhinovirus bronchiolitis.

Bronchiolitis, a viral lower respiratory infection, is the leading cause of infant hospitalization, which is associated with an increased risk for developing asthma later in life. Bronchiolitis can be caused by several respiratory viruses, such as respiratory syncytial virus (RSV), rhinovirus (RV), and others. It can also be caused by a solo infection (e.g., RSV- or RV-only bronchiolitis) or co-infection with two or more viruses. Studies have shown viral etiology-related differences between RSV- and RV-only bronchiolitis in the immune response, human microRNA (miRNA) profiles, and dominance of certain airway microbiome constituents. Here, we identified bacterial small RNAs (sRNAs), the prokaryotic equivalent to eukaryotic miRNAs, that differ between infants of the 35th Multicenter Airway Research Collaboration (MARC-35) cohort with RSV- versus RV-only bronchiolitis. We first derived reference sRNA datasets from cultures of four bacteria known to be associated with bronchiolitis (i.e., Haemophilus influenzae, Moraxella catarrhalis, Moraxella nonliquefaciens, and Streptococcus pneumoniae). Using these reference sRNA datasets, we found several sRNAs associated with RSV- and RV-only bronchiolitis in our human nasal RNA-Seq MARC-35 data. We also determined potential human transcript targets of the bacterial sRNAs and compared expression of the sRNAs between RSV- and RV-only cases. sRNAs are known to downregulate their mRNA target, we found that, compared to those associated with RV-only bronchiolitis, sRNAs associated with RSV-only bronchiolitis may relatively activate the IL-6 and IL-8 pathways and relatively inhibit the IL-17A pathway. These data support that bacteria may be contributing to inflammation differences seen in RSV- and RV-only bronchiolitis, and for the first time indicate that the potential mechanism in doing so may be through bacterial sRNAs.

Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA.

Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called "one-label, one-technique" approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2'-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.

Genetic disruption of the bacterial raiA motif noncoding RNA causes defects in sporulation and aggregation.

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the "raiA motif" has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor.

Pseudomonas aeruginosa outer membrane vesicle-packed sRNAs can enter host cells and regulate innate immune responses.

Bacterial outer membrane vesicles (OMVs) can package and deliver virulence factors into host cells, which is an important mechanism mediating host-pathogen interactions. It has been reported that small RNAs (sRNAs) can be packed into OMVs with varying relative abundance, which might affect the function and/or stability of host mRNAs. In this study, we used OptiPrep density gradient ultra-high-speed centrifugation to purify OMVs from Pseudomonas aeruginosa. Next, the sequences and abundance of sRNAs were detected by using Small RNA-Seq. In particular, sRNA4518698, sRNA2316613 and sRNA809738 were the three most abundant sRNAs in OMVs, which are all fragments of P. aeruginosa non-coding RNAs. sRNAs were shielded within the interior of OMVs and remained resistant to external RNase cleavage. The miRanda and RNAhybrid analysis demonstrated that those sRNAs could target a large number of host mRNAs, which were enriched in host immune responses by the functions of GO and KEGG enrichment. Experimentally, we demonstrated that the transfection of synthetic sRNA4518698, sRNA2316613, or sRNA809738 could reduce the expression of innate immune response genes in RAW264.7 cells. Together, we demonstrated that P. aeruginosa OMVs sRNAs can regulate innate immune responses. This study uncovered a mechanism in which the OMVs regulate host responses by transferring bacterial sRNAs.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as "functional amyloids". In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

Agnodice: indexing experimentally supported bacterial sRNA-RNA interactions.

In the last decade, the immense growth in the field of bacterial small RNAs (sRNAs), along with the biotechnological breakthroughs in Deep Sequencing permitted the deeper understanding of sRNA-RNA interactions. However, microbiology is currently lacking a thoroughly curated collection of this rapidly expanding universe. We present Agnodice (https://dianalab.e-ce.uth.gr/agnodice), our effort to systematically catalog and annotate experimentally supported bacterial sRNA-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Deep Sequencing interactome identification techniques. It comprises 39,600 entries which are annotated at strain-level resolution and pertain to 399 sRNAs and 12,137 target RNAs identified in 71 bacterial strains. The database content is exclusively experimentally supported, incorporating interactions derived via low yield as well as state-of-the-art high-throughput methods. The entire content of the database is freely accessible and can be directly downloaded for further analysis. Agnodice will serve as a valuable source, enabling microbiologists to form novel hypotheses, design/identify novel sRNA-based drug targets, and explore the therapeutic potential of microbiomes from the perspective of small regulatory RNAs.IMPORTANCEAgnodice (https://dianalab.e-ce.uth.gr/agnodice) is an effort to systematically catalog and annotate experimentally supported bacterial small RNA (sRNA)-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Next Generation Sequencing interactome identification techniques.

Direct and indirect control of Rho-dependent transcription termination by the Escherichia coli lysC riboswitch.

Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron.

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.