Structural insights into RNA-mediated transcription regulation in bacteria.

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Hierarchy of RNA functional dynamics.

RNA dynamics play a fundamental role in many cellular functions. However, there is no general framework to describe these complex processes, which typically consist of many structural maneuvers that occur over timescales ranging from picoseconds to seconds. Here, we classify RNA dynamics into distinct modes representing transitions between basins on a hierarchical free-energy landscape. These transitions include large-scale secondary-structural transitions at >0.1-s timescales, base-pair/tertiary dynamics at microsecond-to-millisecond timescales, stacking dynamics at timescales ranging from nanoseconds to microseconds, and other "jittering" motions at timescales ranging from picoseconds to nanoseconds. We review various modes within these three different tiers, the different mechanisms by which they are used to regulate function, and how they can be coupled together to achieve greater functional complexity.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

A Repeat-Associated Small RNA Controls the Major Virulence Factors of Helicobacter pylori.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.

Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis.

Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E.

Substrate Recognition and Autoinhibition in the Central Ribonuclease RNase E.

The endoribonuclease RNase E is a principal factor in RNA turnover and processing that helps to exercise fine control of gene expression in bacteria. While its catalytic activity can be strongly influenced by the chemical identity of the 5' end of RNA substrates, the enzyme can also cleave numerous substrates irrespective of the chemistry of their 5' ends through a mechanism that has remained largely unexplained. We report structural and functional data illuminating details of both operational modes. Our crystal structure of RNase E in complex with the sRNA RprA reveals a duplex recognition site that saddles an inter-protomer surface to help present substrates for cleavage. Our data also reveal an autoinhibitory pocket that modulates the overall activity of the ribonuclease. Taking these findings together, we propose how RNase E uses versatile modes of RNA recognition to achieve optimal activity and specificity.

Regulation by 3'-Untranslated Regions.

3'-untranslated regions (3'-UTRs) are the noncoding parts of mRNAs. Compared to yeast, in humans, median 3'-UTR length has expanded approximately tenfold alongside an increased generation of alternative 3'-UTR isoforms. In contrast, the number of coding genes, as well as coding region length, has remained similar. This suggests an important role for 3'-UTRs in the biology of higher organisms. 3'-UTRs are best known to regulate diverse fates of mRNAs, including degradation, translation, and localization, but they can also function like long noncoding or small RNAs, as has been shown for whole 3'-UTRs as well as for cleaved fragments. Furthermore, 3'-UTRs determine the fate of proteins through the regulation of protein-protein interactions. They facilitate cotranslational protein complex formation, which establishes a role for 3'-UTRs as evolved eukaryotic operons. Whereas bacterial operons promote the interaction of two subunits, 3'-UTRs enable the formation of protein complexes with diverse compositions. All of these 3'-UTR functions are accomplished by effector proteins that are recruited by RNA-binding proteins that bind to 3'-UTR cis-elements. In summary, 3'-UTRs seem to be major players in gene regulation that enable local functions, compartmentalization, and cooperativity, which makes them important tools for the regulation of phenotypic diversity of higher organisms.

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli.

In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.

Stem-Loop Structures within mRNA Coding Sequences Activate Translation Initiation and Mediate Control by Small Regulatory RNAs.

Initiation is the rate-limiting step of translation, and in bacteria, mRNA secondary structure has been extensively reported as limiting the efficiency of translation by occluding the ribosome-binding site. In striking contrast with this inhibitory effect, we report here that stem-loop structures located within coding sequences instead activate translation initiation of the Escherichia coli fepA and bamA mRNAs involved in iron acquisition and outer membrane proteins assembly, respectively. Both structures promote ribosome binding in vitro, independently of their nucleotide sequence. Moreover, two small regulatory RNAs, OmrA and OmrB, base pair to and most likely disrupt the fepA stem-loop structure, thereby repressing FepA synthesis. By expanding our understanding of how mRNA cis-acting elements regulate translation, these data challenge the widespread view of mRNA secondary structures as translation inhibitors and show that translation-activating elements embedded in coding sequences can be targeted by small RNAs to inhibit gene expression.

The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA that differentially regulates gene expression across both genomes.

Satellite viruses are present across all domains of life, defined as subviral parasites that require infection by another virus for satellite progeny production. Phage satellites exhibit various regulatory mechanisms to manipulate phage gene expression to the benefit of the satellite, redirecting resources from the phage to the satellite, and often inhibiting phage progeny production. While small RNAs (sRNAs) are well documented as regulators of prokaryotic gene expression, they have not been shown to play a regulatory role in satellite-phage conflicts. Vibrio cholerae encodes the phage inducible chromosomal island-like element (PLE), a phage satellite, to defend itself against the lytic phage ICP1. Here, we use Hi-GRIL-seq to identify a complex RNA-RNA interactome between PLE and ICP1. Both inter- and intragenome RNA interactions were detected, headlined by the PLE sRNA, SviR. SviR is involved in regulating both PLE and ICP1 gene expression uniquely, decreasing ICP1 target translation and affecting PLE transcripts. The striking conservation of SviR across all known PLEs suggests the sRNA is deeply rooted in the PLE-ICP1 conflict and implicates sRNAs as unidentified regulators of gene expression in phage-satellite interactions.

Molecular basis of FAAH-OUT-associated human pain insensitivity.

Chronic pain affects millions of people worldwide and new treatments are needed urgently. One way to identify novel analgesic strategies is to understand the biological dysfunctions that lead to human inherited pain insensitivity disorders. Here we report how the recently discovered brain and dorsal root ganglia-expressed FAAH-OUT long non-coding RNA (lncRNA) gene, which was found from studying a pain-insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses in patient-derived cells we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed. Given that FAAH is a potential target for the treatment of pain, anxiety, depression and other neurological disorders, this new understanding of the regulatory role of the FAAH-OUT gene provides a platform for the development of future gene and small molecule therapies.

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.

JustRNA: a database of plant long noncoding RNA expression profiles and functional network.

Long noncoding RNAs (lncRNAs) are regulatory RNAs involved in numerous biological processes. Many plant lncRNAs have been identified, but their regulatory mechanisms remain largely unknown. A resource that enables the investigation of lncRNA activity under various conditions is required because the co-expression between lncRNAs and protein-coding genes may reveal the effects of lncRNAs. This study developed JustRNA, an expression profiling resource for plant lncRNAs. The platform currently contains 1 088 565 lncRNA annotations for 80 plant species. In addition, it includes 3692 RNA-seq samples derived from 825 conditions in six model plants. Functional network reconstruction provides insight into the regulatory roles of lncRNAs. Genomic association analysis and microRNA target prediction can be employed to depict potential interactions with nearby genes and microRNAs, respectively. Subsequent co-expression analysis can be employed to strengthen confidence in the interactions among genes. Chromatin immunoprecipitation sequencing data of transcription factors and histone modifications were integrated into the JustRNA platform to identify the transcriptional regulation of lncRNAs in several plant species. The JustRNA platform provides researchers with valuable insight into the regulatory mechanisms of plant lncRNAs. JustRNA is a free platform that can be accessed at http://JustRNA.itps.ncku.edu.tw.

Novel molecular mechanisms of doxorubicin cardiotoxicity: latest leading-edge advances and clinical implications.

Doxorubicin (Dox) is among the most widely used cancer chemotherapeutic drugs. The clinical use of Dox is, however, limited due to its cardiotoxicity. Studies over the past several decades have suggested various mechanisms of Dox-induced cardiotoxicity (DIC). Among them are oxidative stress, topoisomerase inhibition, and mitochondrial damage. Several novel molecular targets and signaling pathways underlying DIC have emerged over the past few years. The most notable advances include discovery of ferroptosis as a major form of cell death in Dox cytotoxicity, and elucidation of the involvement of cardiogenetics and regulatory RNAs as well as multiple other targets in DIC. In this review, we discuss these advances, focusing on latest cutting-edge research discoveries from mechanistic studies reported in influential journals rather than surveying all research studies available in the literature.

Pathogen regulatory RNA usage enables chronic infections, T-cell exhaustion and accelerated T-cell exhaustion.

Pathogens evade or disable cellular immune defenses using regulatory ribonucleic acids (RNAs), including microRNAs and long non-coding RNAs. Pathogenic usage of regulatory RNA enables chronic infections. Chronic infections, using host regulatory RNAs and/or creating pathogenic regulatory RNAs against cellular defenses, can cause T-cell exhaustion and latent pathogen reactivations. Concurrent pathogen infections of cells enable several possibilities. A first pathogen can cause an accelerated T-cell exhaustion for a second pathogen cellular infection. Accelerated T-cell exhaustion for the second pathogen weakens T-cell targeting of the second pathogen and enables a first-time infection by the second pathogen to replicate quickly and extensively. This can induce a large antibody population, which may be inadequately targeted against the second pathogen. Accelerated T-cell exhaustion can explain the relatively short median and average times from diagnosis to mortality in some viral epidemics, e.g., COVID-19, where the second pathogen can lethally overwhelm individuals' immune defenses. Alternatively, if an individual survives, the second pathogen could induce a very high titer of antigen-antibody immune complexes. If the antigen-antibody immune complex titer quickly becomes very high, it can exceed the immune system's phagocytic capability in immuno-deficient individuals, resulting in a Type III hypersensitivity immune reaction. Accelerated T-cell exhaustion in immuno-deficient individuals can be a fundamental cause of several hyperinflammatory diseases and autoimmune diseases. This would be possible when impaired follicular helper CD4+ T-cell assistance to germinal center B-cell somatic hypermutation, affinity maturation and isotype switching of antibodies results in high titers of inadequate antibodies, and this initiates a Type III hypersensitivity immune reaction with proteinase releases which express or expose autoantigens.

Small regulatory RNAs: from bench to bedside - a keystone symposia meeting report.

The Keystone Symposium 'Small Regulatory RNAs: From Bench to Bedside' was held in Santa Fe, New Mexico from May 1-4, 2022. The symposium was organized by Frank J. Slack, Jörg Vogel, Ivan Martinez and Karyn Schmidt, and brought together scientists working in noncoding RNA biology, therapeutics, and technologies to address mechanistic questions about small regulatory RNAs and facilitate translation of these findings into clinical applications. The conference addressed four specific aims: Aim 1. Focus on the exciting biology of small regulatory RNAs, highlighting the best current research into the role that small RNAs play in fundamental biological processes; Aim 2. Focus on the latest efforts to harness the power of these RNAs as agents in the fight against disease and provide the basic understanding that will drive the invention of powerful clinical tools; Aim 3. Attract leaders from both academia and industry working in small RNAs to one place for critical discussions that will advance the field and accelerate the bench to bedside use of this technology; Aim 4. Provide a stimulating environment where students, postdoctoral researchers and junior investigators, along with scientists from Biotechnology and Pharmaceutical companies specializing in small regulatory RNAs, can present and discuss their research with the best minds in the field.

Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

Expression of the Group A Streptococcus Fibrinogen-Binding Protein Mrp Is Negatively Regulated by the Small Regulatory RNA FasX.

The group A Streptococcus (GAS; Streptococcus pyogenes) causes an elaborate array of human diseases. In part, such variability in disease potential is a consequence of GAS manipulating the expression of a catalogue of virulence factors, with regulation occurring at both the transcriptional and posttranscriptional levels. The GAS small regulatory RNA (sRNA) FasX contributes to this regulatory activity, enhancing expression of the thrombolytic agent streptokinase, and reducing expression of collagen (pili) and fibronectin (PrtF1 and PrtF2) -binding adhesins. Here, we expand insight into the regulatory targets of FasX by identifying the M-related protein (Mrp), a fibrinogen-binding adhesin with anti-phagocytic activity, as a negatively-regulated target of FasX. Importantly, investigation of the consequences of FasX-mediated regulation led to the discovery that FasX is a major positive regulator of GAS survival and proliferation in non-immune whole human blood, with a 30-fold difference in GAS cell numbers between a fasX mutant strain and isogenic parental and complemented mutant strains. No difference in cell numbers were observed when these strains were grown in human serum, consistent with the protective phenotype associated with FasX occurring due to the inhibition of cell (e.g., neutrophil) - mediated GAS killing. The FasX-regulated factor/s responsible for the blood survival phenotype remain to be defined. In summary, we expand the known FasX regulon and identify a new phenotype associated with the regulatory activity of this key GAS sRNA. IMPORTANCE Small regulatory RNAs (sRNAs) represent a major class of regulatory molecule that promotes the ability of the group A Streptococcus (GAS) and other pathogens to regulate virulence factor expression. Despite FasX being the best-described sRNA in GAS, there remains much to be learned. Here, we highlight the importance of FasX, identifying for the first time that the loss of this sRNA results in a major reduction in the ability of GAS to survive in human blood, a phenotype critical to the ability of this human-specific pathogen to cause severe invasive infections. We also identified a novel regulatory target of FasX, thereby expanding the known regulon of this key sRNA.

The small regulatory RNA Lpr10 regulates the expression of RpoS in Legionella pneumophila.

Legionella pneumophila (Lp) is a waterborne bacterium able to infect human alveolar macrophages, causing Legionnaires' disease. Lp can survive for several months in water, while searching for host cells to grow in, such as ciliates and amoeba. In Lp, the sigma factor RpoS is essential for survival in water. A previous transcriptomic study showed that RpoS positively regulates the small regulatory RNA Lpr10. In the present study, deletion of lpr10 results in an increased survival of Lp in water. Microarray analysis and RT-qPCR revealed that Lpr10 negatively regulates the expression of RpoS in the postexponential phase. Electrophoretic mobility shift assay and in-line probing showed that Lpr10 binds to a region upstream of the previously identified transcription start sites (TSS) of rpoS. A third putative transcription start site was identified by primer extension analysis, upstream of the Lpr10 binding site. In addition, nlpD TSS produces a polycistronic mRNA including the downstream gene rpoS, indicating a fourth TSS for rpoS. Our results suggest that the transcripts from the third and fourth TSS are negatively regulated by the Lpr10 sRNA. Therefore, we propose that Lpr10 is involved in a negative regulatory feedback loop to maintain expression of RpoS to an optimal level.

6S RNA-Dependent Susceptibility to RNA Polymerase Inhibitors.

Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate a wide range of pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor. This effect applied to rifabutin and fidaxomicin, two other RNAP-targeting antibiotics. 6S RNA is highly conserved in bacteria, and its absence in two other major pathogens, Salmonella enterica and Clostridioides difficile, also impaired susceptibility to RNAP inhibitors. In S. aureus, 6S RNA is produced from an autonomous gene and accumulates in stationary phase. In contrast to what was reported for Escherichia coli, S. aureus 6S RNA does not appear to play a critical role in the transition from exponential to stationary phase but affects σB-regulated expression in prolonged stationary phase. Nevertheless, its protective effect against rifampicin is independent of alternative sigma factor σB activity. Our results suggest that 6S RNA helps maintain RNAP-σA integrity in S. aureus, which could in turn help bacteria withstand low concentrations of RNAP inhibitors.