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Regulation by Small RNAs in Bacteria: Expanding Frontiers
Abstract
Research on the discovery and characterization of small, regulatory RNAs in bacteria has exploded in recent years. These sRNAs act by base pairing with target mRNAs with which they share limited or extended complementarity, or by modulating protein activity, in some cases by mimicking other nucleic acids. Mechanistic insights into how sRNAs bind mRNAs and proteins, how they compete with each other, and how they interface with ribonucleases are active areas of discovery. Current work also has begun to illuminate how sRNAs modulate expression of distinct regulons and key transcription factors, thus integrating sRNA activity into extensive regulatory networks. In addition, the application of RNA deep sequencing has led to reports of hundreds of additional sRNA candidates in a wide swath of bacterial species. Most importantly, recent studies have served to clarify the abundance of remaining questions about how, when, and why sRNA-mediated regulation is of such importance to bacterial lifestyles.
... ncRNAs such as riboswitches, T boxes, and sRNAs are the RNA molecules that play an important role in regulation of gene expression. sRNAs were first reported in Escherichia coli and recognized as key transcriptional regulators [9], due to their swift response to modulate mechanisms in the bacteria [10]. Studies show that ncRNAs regulate biofilm formation in bacteria such as P. aeruginosa [11], B. cepacia complex [11], E. coli [12] and S. enterica Typhi [13]. ...
... Biofilms are complex multilayer structures of bacterial cells where the initial layers in contact with the metal perpetuate anoxic ambience, promoting growth of ncRNAs such as riboswitches, T boxes, and sRNAs are the RNA molecules that play an important role in regulation of gene expression. sRNAs were first reported in Escherichia coli and recognized as key transcriptional regulators [9], due to their swift response to modulate mechanisms in the bacteria [10]. Studies show that ncRNAs regulate biofilm formation in bacteria such as P. aeruginosa [11], B. cepacia complex [11], E. coli [12] and S. enterica Typhi [13]. ...
Noncoding RNAs (ncRNAs) play key roles in the regulation of important pathways, including cellular growth, stress management, signaling, and biofilm formation. Sulfate-reducing bacteria (SRB) contribute to huge economic losses causing microbial-induced corrosion through biofilms on metal surfaces. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation. This study aimed to identify ncRNAs in the genome of a model SRB, Oleidesulfovibrio alaskensis G20 (OA G20). Three in silico approaches revealed genome-wide distribution of 37 ncRNAs excluding tRNAs in the OA G20. These ncRNAs belonged to 18 different Rfam families. This study identified riboswitches, sRNAs, RNP, and SRP. The analysis revealed that these ncRNAs could play key roles in the regulation of several pathways of biosynthesis and transport involved in biofilm formation by OA G20. Three sRNAs, Pseudomonas P10, Hammerhead type II, and sX4, which were found in OA G20, are rare and their roles have not been determined in SRB. These results suggest that applying various computational methods could enrich the results and lead to the discovery of additional novel ncRNAs, which could lead to understanding the “rules of life of OA G20” during biofilm formation.
... Bacteria encounter a wide range of pressures in their natural environment such as nutrient availability, oxidative stress, and presence of antimicrobials. Small regulatory RNAs (sRNAs) aid in the adaptation to these conditions by enabling bacteria to swiftly transition between different physiological states (1,2). These regulatory RNAs also allow bacteria to efficiently allocate resources and maximize their survival in diverse environmental niches. ...
... The hybridization of sRNAs may alter mRNA translation directly by affecting ribosome accessibility or indirectly by modifying mRNA stability (3,4). An important aspect of sRNA-based gene regulation is that it occurs through imperfect base-pairing interactions that allow a single sRNA to act with regulatory plasticity, targeting genes across diverse pathways, linking different biological processes, and facilitating connections within unique cellular networks (2,5). ...
Small regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses in Escherichia coli and other bacteria. sRNAs act post-transcriptionally on target mRNA through an imperfect base pairing mechanism to regulate downstream protein expression. The imperfect base pairing allows a single sRNA to bind and regulate a variety mRNA targets which can form intricate regulatory networks that connect different physiological processes for the cell’s response. Upon exposure to antimicrobials and superoxide generating agents, the MicF sRNA in E. coli has been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes in E. coli that are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter-gene fusion, we have validated two novel targets of MicF regulation: SeqA, a negative modulator of DNA replication, and ObgE, a GTPase crucial for chromosome partitioning. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicF’s conserved 5’ seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation.
... tRNAs and rRNAs) ( 1 ,2 ) to the regulation of gene expression (e.g. sRNAs and riboswitches) (3)(4)(5)(6). In addition to these ancient and widespread classes of ncRNAs involved in translation and gene regulation, numerous other ncRNA classes are known to be broadly distributed and carry out other important functions. ...
Structured noncoding RNAs (ncRNAs) contribute to many important cellular processes involving chemical catalysis, molecular recognition and gene regulation. Few ncRNA classes are broadly distributed among organisms from all three domains of life, but the list of rarer classes that exhibit surprisingly diverse functions is growing. We previously developed a computational pipeline that enables the near-comprehensive identification of structured ncRNAs expressed from individual bacterial genomes. The regions between protein coding genes are first sorted based on length and the fraction of guanosine and cytidine nucleotides. Long, GC-rich intergenic regions are then examined for sequence and structural similarity to other bacterial genomes. Herein, we describe the implementation of this pipeline on 50 bacterial genomes from varied phyla. More than 4700 candidate intergenic regions with the desired characteristics were identified, which yielded 44 novel riboswitch candidates and numerous other putative ncRNA motifs. Although experimental validation studies have yet to be conducted, this rate of riboswitch candidate discovery is consistent with predictions that many hundreds of novel riboswitch classes remain to be discovered among the bacterial species whose genomes have already been sequenced. Thus, many thousands of additional novel ncRNA classes likely remain to be discovered in the bacterial domain of life.
... permitir el ingreso de la subunidad ribosomal 30s[31,32,[55][56][57][58][59][60][61][62]. La Figura I.2 presenta un esquema de este mecanismo. ...
The Csr protein family is present in 75% of bacterial species, and it is estimated that 15% of the genes encoded in Escherichia coli are regulated by them. Their biological role is associated with their ability to bind to the 5' untranslated regions (UTRs) of messenger RNA (mRNA), thereby modifying their translation rate. Among the target mRNAs of RsmE, the hcnA gene stands out, which encodes one of the subunits of the enzyme responsible for synthesizing HCN in Pseudomonas protegens. This metabolite acts as a natural inhibitor against soil pathogens, providing natural protection to plants whose roots it inhabits. Homologs of the Csr family in the Pseudomonas genus are named with the prefix "Rsm." They prevent the 30s ribosomal subunit's accessibility to the target gene, so the presence of the protein implies repression in mRNA expression. The Csr/Rsm family is, in turn, regulated by non-coding and short sequence RNA molecules (sRNA), capable of capturing the protein and releasing the gene they regulate. These sRNAs have the same consensus binding sequence as the mRNAs captured by Csr/Rsm, acting as competitive inhibitors. Among these sRNAs, RsmZ is the only one whose structure has been experimentally characterized. In those studies, it was determined that RsmE cooperatively binds to different binding pockets of RsmZ in a defined order. Conversely, previous experimental studies show that RsmE binds to hcnA in an anticooperative manner. The goal of this thesis is to explain these aspects of the system. For this, an analysis based on Molecular Dynamics (MD) simulations is presented, exploring the conformational diversity of both RsmZ and the RsmE-RsmZ and RsmE-hcnA complexes. The results reveal a defined pattern of sequential exposure of different binding motifs of RsmZ caused by the capture of successive units of RsmE. Together, these simulations provide a simple and consistent explanation for the observed order of binding, the molecular bases of which had not been unveiled until now. Additionally, structural models for partially occupied RsmE-RsmZ complexes that had not been described experimentally at the time of this thesis are presented. On the other hand, Umbrella Sampling simulations were implemented to describe the binding mechanism between RsmE and the RsmZ binding site located in a single-stranded region. The study aimed to elucidate the most relevant interactions of the process, estimating the energy involved in the binding event. It was observed that the studied motif of RsmZ adopts a stem-loop-like structure during the event, and to reach that conformation, it is necessary for the single-stranded segment to contain at least nine nucleotides. Also, through Principal Component Analysis (PCA) of both molecules in their free form, it was determined that their main collective coordinates tend to generate conformations conducive to binding, strongly suggesting that the flexibility of the single-stranded region of RsmZ crucially affects its ability to capture RsmE. Finally, preliminary results are presented to elucidate the binding mechanism between RsmE and the hcnA gene. The Umbrella Sampling simulations presented faithfully reproduce the experimental results and can, therefore, be applied to their rationalization. In summary, this work presents the first reported simulations for both the post-transcriptional regulation system RsmE-RsmZ and the RsmE-hcnA complex, adding to the limited background of MD techniques applied to the study of sRNA molecules.
... It could bind to specific small molecules (ligands) and regulate gene expression through changes in secondary structure of the mRNAs and thus the binding of the ribosomes [3,4]. sRNAs are typically trans-encoded regulatory RNAs with an average length of 50~300 nt [5] and interact with multiple target mRNAs by imperfect base pairing, causing mRNA degradation or translation blocking. Antisense RNAs engage in extensive base-pairing interactions with the complementary mRNA, as they are transcribed from the DNA strand opposite to that encoding the mRNA and can span from ten to thousands of nucleotides in length [6]. ...
Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels, including small RNAs (sRNAs), riboswitches, and antisense RNAs. Notably, the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription, post-transcription or translation processes. Leveraging and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic biology. While previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica, this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria, notably Bacillus subtilis. Furthermore, we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology, employing B. subtilis as a foundational chassis.
... The present findings revealed that deletion of snc251 affected multiple cellular processes and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress (Fig. 5g). sRNAs are typically 50-400 nt long (14,31) and act by base pairing with target mRNAs or by modulating protein activity (32). Both RACE and Northern blot analysis showed that sRNA SNC251 is 263 nt in length ( Fig. 3a and b). ...
Drought and salinity are ubiquitous environmental factors that pose hyperosmotic threats to microorganisms and impair their efficiency in performing environmental functions. However, bacteria have developed various responses and regulatory systems to cope with these abiotic challenges. Posttranscriptional regulation plays vital roles in regulating gene expression and cellular homeostasis, as hyperosmotic stress conditions can lead to the induction of specific small RNA molecules (sRNAs) that participate in stress response regulation. Here, we report a candidate functional sRNA landscape of Sphingomonas melonis TY under hyperosmotic stress, and 18 sRNAs were found with a clear response to hyperosmotic stress. These findings will help in the comprehensive analysis of sRNA regulation in Sphingomonas species. Weighted correlation network analysis revealed a 263 nucleotide sRNA, SNC251, which was transcribed from its own promoter and showed the most significant correlation with hyperosmotic response factors. Deletion of snc251 affected biofilm formation and multiple cellular processes, including ribosome-related pathways, aromatic compound degradation, and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress. Two genes involved in the TonB system were further verified to be activated by SNC251, which also indicated that SNC251 is a trans-acting sRNA. Briefly, this research reports a landscape of sRNAs participating in the hyperosmotic stress response in S. melonis and reveals a novel sRNA, SNC251, which contributes to the S. melonis TY biofilm formation and thus enhances its hyperosmotic stress response ability.
IMPORTANCE
Sphingomonas species play a vital role in plant defense and pollutant degradation and survive extensively under drought or salinity. Previous studies have focused on the transcriptional and translational responses of Sphingomonas under hyperosmotic stress, but the posttranscriptional regulation of small RNA molecules (sRNAs) is also crucial for quickly modulating cellular processes to adapt dynamically to osmotic environments. In addition, the current knowledge of sRNAs in Sphingomonas is extremely scarce. This research revealed a novel sRNA landscape of Sphingomonas melonis and will greatly enhance our understanding of sRNAs’ acting mechanisms in the hyperosmotic stress response.
... In addition to sigma factors, recent studies have discovered that small regulatory RNAs (sRNAs) have been widely studied due to their meaningful regulatory roles in bacteria especially on virulence genes (Lee and Gottesman, 2016;Gao et al., 2017;Ahmed et al., 2019). Similar to transcription factors, sRNAs can interfere or increase ribosome binding, strengthen or weaken mRNA stability, and some even can control the activity of the protein (Storz et al., 2011). Environmental stresses reveal sRNAs to coordinate the adaption processes of a bacterium (Barnhill et al., 2019). ...
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne pathogen which is frequently used as the reference strain for Salmonella. Investigating the sigma factor network and protomers is crucial to understand the genomic and transcriptomic properties of the bacterium. Its promoters were identified using various methods such as dRNA-seq, ChIP-chip, or ChIP-Seq. However, validation using ChIP-exo, which exhibits higher-resolution performance compared to conventional ChIP, has not been conducted to date. In this study, using the representative strain S. Typhimurium LT2 (LT2), the ChIP-exo experiment was conducted to accurately determine the binding sites of catalytic RNA polymerase subunit RpoB and major sigma factors (RpoD, RpoN, RpoS, and RpoE) during exponential phase. Integrated with the results of RNA-Seq, promoters and sigmulons for the sigma factors and their association with RpoB have been discovered. Notably, the overlapping regions among binding sites of each alternative sigma factor were found. Furthermore, comparative analysis with Escherichia coli str. K-12 substr. MG1655 (MG1655) revealed conserved binding sites of RpoD and RpoN across different species. In the case of small RNAs (sRNAs), 50 sRNAs observed their expression during the exponential growth of LT2. Collectively, the integration of ChIP-exo and RNA-Seq enables genome-scale promoter mapping with high resolution and facilitates the characterization of binding events of alternative sigma factors, enabling a comprehensive understanding of the bacterial sigma factor network and condition-specific active promoters.
... Given the strong regulatory link between the MavR sRNA and the production of the MurF protein ( Figure 3), we set out to identify the mechanism of this regulatory event. The predicted secondary structure of MavR revealed several stem-loop structures characteristic of bacterial sRNAs (Storz et al., 2011), and one of the predicted loops contains a 6-nucleotide sequence (5′-CUCCUC) with complementarity to the ribosome-binding site found just upstream of the coding sequence in the MurF mRNA (5′-GAGGAG) ( Figure S7). We predicted that this 6-nucleotide sequence mediates the negative regulation of MurF production, and to test this prediction, we performed site-directed mutagenesis to alter the CUCCUC sequence in MavR. ...
Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post‐transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF‐ and virulence‐regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6‐nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.
... Furthermore, lineage-specific SNPs was shown to alter the transcriptome of Indo-Oceanic and East-Asian lineages, resulting in differential gene expression with unique transcriptional regulatory changes [43]. Transcriptome is tightly regulated by the non-coding sRNAs in bacterial species [79], including M. tuberculosis [23,24]. Small noncoding RNAs (sRNAs) make up the largest group of regulatory RNAs. ...
Tuberculosis has been a public health crisis since the 1900, which has caused the highest mortalities due to a single bacterial infection worldwide, that was recently further complicated by the Coronavirus disease 2019 pandemic. The causative agent of Tuberculosis, Mycobacterium tuberculosis, belongs to a genetically well-characterized family of strains known as the Mycobacterium tuberculosis complex, which has complicated progress made towards eradicating Tuberculosis due to pathogen-specific phenotypic differences in the members of this complex. Mycobacterium tuberculosis complex strains are genetically diverse human- and animal-adapted pathogens belonging to 7 lineages (Indo-Oceanic, East-Asian, East-African Indian, Euro-American, M. africanum West Africa 1, M. africanum West Africa 2 and Ethopia), respectively and the recently identified Lineage 8 and M. africanum Lineage 9. Genomic studies have revealed that Mycobacterium tuberculosis complex members are ∼99 % similar, however, due to selective pressure and adaptation to human host, they are prone to mutations that have resulted in development of drug resistance and phenotypic heterogeneity that impact strain virulence. Furthermore, members of the Mycobacterium tuberculosis complex have preferred geographic locations and possess unique phenotypic characteristics that is linked to their pathogenicity. Due to the recent advances in development next generation sequencing platforms, several studies have revealed epigenetic changes in genomic regions combined with “unique” gene regulatory mechanisms through non-coding RNAs that are responsible for strain-specific behaviour on in vitro and in vivo infection models. The current review provides up to date epigenetic patterns, gene regulation through non-coding RNAs, together with implications of these mechanisms in down-stream proteome and metabolome, which may be responsible for “unique” responses to infection by members of the Mycobacterium tuberculosis complex. Understanding lineage-specific molecular mechanisms during infection may provide novel drug targets and disease control measures towards World Health organization END-TB strategy.
... While most studies performed in the last decades have led to the idea that sRNAs regulate gene expression at the post-transcriptional level (51)(52)(53), it has recently been shown that sRNAs can also operate cotranscriptionally by controlling Rho transcription termination (54)(55)(56). For ChiX, Rho transcription termination is indirectly controlled through the modulation of translation initiation in Salmonella enterica (57). ...
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
... They have been found to serve as posttranscriptional regulators that affect almost every field of bacterial physiology, including quorum sensing, environmental stimulus-response, and biofilm formation (Bossi and Figueroa-Bossi, 2016;Carroll et al., 2016;Lei et al., 2019;Merritt et al., 2014). Many sRNAs are found to be formed through interactions with host cells and, in turn, affect their viability in the host (Papenfort and Vogel, 2010;Storz et al., 2011). Therefore, bacterial sRNAs are considered novel biomarkers for oral microbiota-associated cancer therapy. ...
The oral microbiota is a vital part of the human microbiota that functions in various physiological processes and is highly relevant to cancers and other diseases. With the alterations of host immune competence, the homeostatic balance existing between the oral microbiota and host may be disturbed and result in the development of diseases. Numerous observations have suggested that small RNAs are key regulators of bacterial pathogenesis and bacteria-host interactions. Further, bacterial small RNAs are considered to be promising biomarkers for the development of novel, and efficacious therapies for oral dysbiosis. Mechanistic insights into how oral pathogens communicate with other bacteria or host cells in oral cancers via small RNAs are hot topics of research. Current studies also have begun to elucidate the key role of oral microbiota in the development of systemic diseases. This article discusses existing findings and nascent mechanisms governing the small RNA-based interactions between oral microbiota and associated diseases. The knowledge of such interactions is key in planning approaches to reverse dysbiosis to achieve health.
... Moreover, the discovery of a variety of novel regulatory non-coding RNAs through RNA-seq has rekindled interest in RNase III. Non-coding regulatory RNAs are part of the riboregulation network and were not only described in bacteria (in bacteria called sRNAs) but also in numerous other organisms (reviewed in Jørgensen et al., 2020;Mahendran et al., 2022;Papenfort & Melamed, 2023;Storz et al., 2011). sRNAs can exert their regulatory effects on gene expression by specific base pairing with their target mRNAs, and thereby form RNA-RNA duplexes, which are often recognised by RNase III as a substrate (Lioliou et al., 2012;McKellar et al., 2022;Mediati et al., 2022). ...
RNase III is a dsRNA‐specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α‐proteobacterium Rhodobacter sphaeroides. RNA‐seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type‐like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum‐sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment‐protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III‐mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.
... Small RNAs (sRNAs) are critical regulatory elements that have been a focus of investigation in eukaryotes but remain relatively unexplored in prokaryotes. They are short (typically 18-500 nt), non-coding transcripts that can function as either enhanc ers or repressors of gene expression, often via binding to their complementary mRNA targets (28)(29)(30)(31). Importantly, sRNAs can mediate prompt and energetically low-cost gene regulation post-transcriptionally (32)(33)(34)(35)(36). ...
Dental caries is among the most prevalent chronic diseases worldwide. Streptococcus mutans , the chief causative agent of caries, uses a 25-kDa manganese-dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression, and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we focused our attention on 18–50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of RNA sequencing revealed 19 sRNAs in S. mutans, which were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 10 sRNAs that were differentially expressed in UA159 cells grown in the presence of low vs high manganese. We describe SmsR1532 and SmsR1785 as SloR- and manganese-responsive sRNAs that are processed from large transcripts and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen.
IMPORTANCE
Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25-kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterized sRNAs that are both SloR and manganese responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen and that can enable the development of an effective anti-caries therapeutic.
... The paradigm shift of the central dogma of the role of RNA in the cell as messenger, transfer or ribosomal RNA has it now implicated in a multitude of processes in regulatory or enzymatic roles (Scott, 2007;Storz et al., 2011;Breaker, 2018). 6S is one such molecule, a master regulator of transcription being highly conserved in most bacterial families (Wehner et al., 2014). ...
The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.
... Despite its importance as a zoonotic pathogen, there remains a significant knowledge gap related to the molecular mechanisms that enable C. jejuni to rapidly adapt to drastically different host environments and result in either commensal carriage or disease of the host. Knowledge regarding post-transcriptional regulation of gene expression in C. jejuni via small non-coding RNAs (sRNAs) has been particularly scarce due to the lack of apparent sRNA homologs in other enteric pathogens, where sRNAs have been shown to be key regulators influencing numerous phenotypes including metabolism, motility, biofilm production, and virulence factors (5)(6)(7)(8)(9)(10)(11). sRNAs function by base pairing to their target mRNAs, typically within the 5´ untranslated regions of the target mRNA; sRNA binding then leads to changes in the stability of the mRNA transcript, affecting translation positively or negatively depending on the location of the interaction (7,9). ...
Small non-coding RNAs (sRNAs) are important players in modulating gene expression in bacterial pathogens, but their functions are largely undetermined in Campylobacter jejuni , an important cause of foodborne gastroenteritis in humans. In this study, we elucidated the functions of sRNA CjNC140 and its interaction with CjNC110, a previously characterized sRNA involved in the regulation of several virulence phenotypes of C. jejuni . Inactivation of CjNC140 increased motility, autoagglutination, L-methionine concentration, autoinducer-2 production, hydrogen peroxide resistance, and early chicken colonization, indicating a primarily inhibitory role of CjNC140 for these phenotypes. Apart from motility, all these effects directly contrasted the previously demonstrated positive regulation by CjNC110, suggesting that CjNC110 and CjNC140 operate in an opposite manner to modulate physiologic processes in C. jejuni . RNAseq and northern blotting further demonstrated that expression of CjNC140 increased in the absence of CjNC110, while expression of CjNC110 decreased in the absence of CjNC140, suggesting a possibility of their direct interaction. Indeed, electrophoretic mobility shift assay demonstrated a direct binding between the two sRNAs via GA- (CjNC110) and CU- (CjNC140) rich stem-loops. Additionally, RNAseq and follow-up experiments identified that CjNC140 positively regulates p19 , which encodes a key iron uptake transporter in Campylobacter . Furthermore, computational analysis revealed both CjNC140 and CjNC110 are highly conserved in C. jejuni, and the predicted secondary structures support CjNC140 as a functional homolog of the iron regulatory sRNA, RyhB. These findings establish CjNC140 and CjNC110 as a key checks-and- balances mechanism in maintaining homeostasis of gene expression and optimizing phenotypes critical for C. jejuni pathobiology.
IMPORTANCE
Gene regulation is critical to all aspects of pathogenesis of bacterial disease, and small non-coding RNAs (sRNAs) represent a new frontier in gene regulation of bacteria. In Campylobacter jejuni , the role of sRNAs remains largely unexplored. Here, we investigate the role of two highly conserved sRNAs, CjNC110 and CjNC140, and demonstrate that CjNC140 displays a primarily inhibitory role in contrast to a primarily activating role for CjNC110 for several key virulence-associated phenotypes. Our results also revealed that the sRNA regulatory pathway is intertwined with the iron uptake system, another virulence mechanism critical for in vivo colonization. These findings open a new direction for understanding C. jejuni pathobiology and identify potential targets for intervention for this major foodborne pathogen.
... Many types of cis-acting RNA regulators are associated with secondary structure changes and premature termination and/or RNA processing within 5′ untranslated regions (UTRs) of mRNAs 12,18 . The cis-acting RNA structures have been shown to change in response to temperature (RNA thermometers), translation of small upstream open reading frames (uORFs), or by binding trans-acting factors, such as metabolites (riboswitches), tRNAs, RNA-binding proteins, or small base-pairing RNAs (sRNAs) [19][20][21][22][23][24] . sRNAs typically base pair with another RNA to elicit a regulatory response and can be transcribed independently or processed from the 5′ or 3′ UTR of mRNA transcripts, tRNAs, or internal to coding sequences 25 . ...
Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. Here, we use several RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi – the etiological agent of Lyme disease. We identify complex gene arrangements and operons, untranslated regions and small RNAs. We predict intrinsic terminators and experimentally test examples of Rho-dependent transcription termination. Remarkably, 63% of RNA 3′ ends map upstream of or internal to open reading frames (ORFs), including genes involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs result from premature termination, processing and regulatory events such as cis-acting regulation. Furthermore, the polyamine spermidine globally influences the generation of truncated mRNAs. Collectively, our findings provide insights into transcription termination and uncover an abundance of potential RNA regulators in B. burgdorferi.
... Small RNAs (sRNAs) are important regulators of various physiological responses in bacteria (Waters and Storz 2009). These noncoding RNAs are usually 50-300 nt long and act as post-transcriptional regulators (Storz et al. 2011). In Escherichia coli, several noncoding sRNAs with regulatory roles in stress response and adaptation to environmental changes have been identified (Gottesman 2004). ...
Small non-coding RNAs are an important class of regulatory RNAs in bacteria, often regulating responses to changes in environmental conditions. OxyS is a 110 nucleotide, stable, trans-encoded small RNA found in Escherichia coli and is induced by an increased concentration of hydrogen peroxide. OxyS has an important regulatory role in cell stress response, affecting the expression of multiple genes. In this work, we investigated the structure of OxyS and the interaction with fhlA mRNA using nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and unbiased molecular dynamics simulations. We determined the secondary structures of isolated stem-loops and confirmed their structural integrity in OxyS. Unexpectedly, stem-loop SL4 was identified in the region that was predicted to be unstructured. Three-dimensional models of OxyS demonstrate that OxyS adopts an extended structure with four solvent-exposed stem-loops, which are available for interaction with other RNAs and proteins. Furthermore, we provide evidence of base pairing between OxyS and fhlA mRNA.
... Over the course of these investigations, the enzymes responsible for writing (catalyzing and modifying nucleotides), reading (recognizing and binding modified nucleotides) and erasing (catalyzing the removal of specific modifications) RNA modifications have also been discovered [6][7][8][9]. Small RNAs, which are a class of noncoding RNAs that are less than 200 nucleotides in length, are widely present in various cell types and tissues [10][11][12]. Over the past 20 years, extensive research has led to their classification on the basis of their size and structural characteristics, as follows: traditional small RNAs, structural small RNAs and derived small RNAs (also called non-canonical small RNAs, Fig. 1 and Table 1) [13]. ...
Small RNAs (also referred to as small noncoding RNAs, sncRNA) are defined as polymeric ribonucleic acid molecules that are less than 200 nucleotides in length and serve a variety of essential functions within cells. Small RNA species include microRNA (miRNA), PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), tRNA-derived small RNA (tsRNA), etc. Current evidence suggest that small RNAs can also have diverse modifications to their nucleotide composition that affect their stability as well as their capacity for nuclear export, and these modifications are relevant to their capacity to drive molecular signaling processes relevant to biogenesis, cell proliferation and differentiation. In this review, we highlight the molecular characteristics and cellular functions of small RNA and their modifications, as well as current techniques for their reliable detection. We also discuss how small RNA modifications may be relevant to the clinical applications for the diagnosis and treatment of human health conditions such as cancer.
... Small RNAs (sRNAs) in bacteria are typically 50-400 nucleotides in length, playing a key role in regulating gene expression [46,47]. In the canonical pathway regulating sRNA-mediated gene expression, sRNAs, often along with the chaperone protein Hfq, positively or negatively regulate the target mRNA expression in response to environmental changes through an incomplete Watson-Crick base pairing [48]. ...
Background:
Dental caries is a chronic, multifactorial and biofilm-mediated oral bacterial infection affecting almost every age group and every geographical region. Streptococcus mutans is considered an important pathogen responsible for the initiation and development of dental caries. It produces exopolysaccharides in situ to promote the colonization of cariogenic bacteria and coordinate dental biofilm development.
Objective:
The understanding of the regulatory mechanism of S. mutans biofilm formation can provide a theoretical basis for the prevention and treatment of caries.
Design:
At present, an increasing number of studies have identified many regulatory systems in S. mutans that regulate biofilm formation, including second messengers (e.g. c-di-AMP, Ap4A), transcription factors (e.g. EpsR, RcrR, StsR, AhrC, FruR), two-component systems (e.g. CovR, VicR), small RNA (including sRNA0426, srn92532, and srn133489), acetylation modifications (e.g. ActG), CRISPR-associated proteins (e.g. Cas3), PTS systems (e.g. EIIAB), quorum-sensing signaling system (e.g. LuxS), enzymes (including Dex, YidC, CopZ, EzrA, lmrB, SprV, RecA, PdxR, MurI) and small-molecule metabolites.
Results:
This review summarizes the recent progress in the molecular regulatory mechanisms of exopolysaccharides synthesis and biofilm formation in S. mutans.
... [3] It had also been shown in the preceding decades that small RNAs are also widely used for gene control in prokaryotes, mainly by antisense mechanisms. [3,51] The seventh anomaly was revealed by the genome sequencing projects at the turn of the century-the lack of scaling of the number of protein-coding genes with developmental complexity, the 'g-value enigma' , [52] thought to be compensated by the power of transcription factor combinatorics [9] and an increase in the numbers of protein isoforms generated by alternative splicing. [53] It should also be noted that, until recently, the identification of protein-coding genes was largely restricted to those greater than 100 codons, automatically excluding many encoding smaller peptides and those with non-canonical start codons. ...
Thomas Kuhn described the progress of science as comprising occasional paradigm shifts separated by interludes of 'normal science'. The paradigm that has held sway since the inception of molecular biology is that genes (mainly) encode proteins. In parallel, theoreticians posited that mutation is random, inferred that most of the genome in complex organisms is non-functional, and asserted that somatic information is not communicated to the germline. However, many anomalies appeared, particularly in plants and animals: the strange genetic phenomena of paramutation and transvection; introns; repetitive sequences; a complex epigenome; lack of scaling of (protein-coding) genes and increase in 'noncoding' sequences with developmental complexity; genetic loci termed 'enhancers' that control spatiotemporal gene expression patterns during development; and a plethora of 'intergenic', overlapping, antisense and intronic transcripts. These observations suggest that the original conception of genetic information was deficient and that most genes in complex organisms specify regulatory RNAs, some of which convey intergenerational information.
... In addition to the two-component regulatory and quorum sensing systems, sRNAs play important roles in the post-transcriptional regulation of various biological processes including the formation of biofilms and the development of biofilm-associated antibiotic resistance [16,[21][22][23][24]. sRNAs interact with the target mRNAs by forming base pairs at or near the ribosome binding sites of the target, which produce either a positive or negative influence on various biological processes, including biofilm development, in the bacterial cells [5,23,[25][26][27][28][29][30][31]. ...
Small RNAs (sRNAs) are post-transcriptional regulators of many biological processes in bacteria, including biofilm formation and antibiotic resistance. The mechanisms by which sRNA regulates the biofilm-specific antibiotic resistance in Acinetobacter baumannii have not yet been reported. This study aimed to investigate the influence of sRNA00203 (53 nucleotides) on biofilm formation, antibiotic susceptibility, and expression of genes associated with biofilm formation and antibiotic resistance. Our results showed that deletion of the sRNA00203-encoding gene substantially decreased the biomass of biofilm by 85%. Deletion of the sRNA00203-encoding gene also reduced the minimum biofilm inhibitory concentration for imipenem and ciprofloxacin by 1,024 and 128 folds, respectively. Besides, knocking out of sRNA00203 significantly downregulated genes involved in biofilm matrix synthesis (pgaB), efflux pump production (novel00738), lipopolysaccharide biosynthesis (novel00626), preprotein translocase subunit (secA), and the CRP transcriptional regulator. Overall, we found that suppression of sRNA00203 in an A. baumannii ST1894 strain impaired biofilm formation and sensitized the biofilm cells to imipenem and ciprofloxacin. As sRNA00203 was found to be conserved in A. baumannii, a therapeutic strategy targeting sRNA00203 may be a potential solution to the treatment of biofilm-associated infections caused by A. baumannii. To the best of our knowledge, this is the first study to show the impact of sRNA00203 on biofilm formation and biofilm-specific antibiotic resistance in A. baumannii.
... Bacterial sRNAs are typically untranslated transcripts, 50 to 500 nucleotides in length. Regulation of target mRNAs by sRNA is achieved through base matching [15]. According to the regulation mode, bacterial sRNAs are divided into transcoded and cis-coded sRNAs. ...
In recent years, the role of bacterial sRNAs in adversity tolerance and pathogens has attracted increasing attention. A great number of virulence-related sRNAs were reported in a variety of human pathogens. However, only a few sRNAs from aquatic pathogens were reported. In our previous study, a novel sRNA, sR158, was identified in Edwardsiella piscicida, an important aquatic pathogen, but its function remains unknown. In the same aquatic pathogen, we also identified a type II TA system, YefM-YoeB, in another study. In the current report, we found that the expression of yefM-yoeB in E. piscicida was regulated by sR158, which is dependent on the RNA chaperon Hfq. The deletion of sR158 reduced bacterial tolerance to oxidation pressure, enhanced bacterial capacity for biofilm formation, increased bacterial adhesion and invasion of host cells and immune tissues, and boosted bacterial general virulence, which are consistent with the effects caused by the deletion of YefM-YoeB. These findings indicate that sR158 participates in the stress resistance and virulence of E. piscicida by regulating YefM-YoeB. Our result is the first report that the type II TA system is regulated by sRNA, which provides new insights into the regulatory role of bacterial sRNA.
Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create ‘superbugs’ that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.
Computational prediction of RNA-RNA interactions (RRI) is a central methodology for the specific investigation of inter-molecular RNA interactions and regulatory effects of non-coding RNAs like eukaryotic microRNAs or prokaryotic small RNAs. Available methods can be classified according to their underlying prediction strategies, each implicating specific capabilities and restrictions often not transparent to the non-expert user. Within this work, we review seven classes of RRI prediction strategies and discuss the advantages and limitations of respective tools, since such knowledge is essential for selecting the right tool in the first place.
Among the RRI prediction strategies, accessibility-based approaches have been shown to provide the most reliable predictions. Here, we describe how IntaRNA, as one of the state-of-the-art accessibility-based tools, can be applied in various use cases for the task of computational RRI prediction. Detailed hands-on examples for individual RRI predictions as well as large-scale target prediction scenarios are provided. We illustrate the flexibility and capabilities of IntaRNA through the examples. Each example is designed using real-life data from the literature and is accompanied by instructions on interpreting the respective results from IntaRNA output. Our use-case driven instructions enable non-expert users to comprehensively understand and utilize IntaRNA’s features for effective RRI predictions.
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also
controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that
OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
The subject of the study is the importance of molecular genetic research methods in the study of the etiopathogenesis of osteomyelitis of the jaws. The purpose of the work is to provide up-to-date information to researchers, dental surgeons, and maxillofacial surgeons on the possibilities of molecular genetic research in identifying bacterial pathogens in osteomyelitis of the jaws, as well as to reflect genetic markers of pathogenicity factors for a number of the main causative agents of the disease. Methodology. International scientific databases PubMed, ScienceDirect, Scopus, Cochrane Collaboration, Elsevier, as well as electronic catalogs eLIBRARY.RU and CyberLeninka.ru were used. Results. A review of publications demonstrated that S. aureus and S. Epidermidis dominate the etiological spectrum of causative agents of bone tissue infections. The participation of these microorganisms is determined by a whole range of pathogenicity factors. Toxins and Panton-Valentine leukocidin (PVL) genes play a major role in the pathogenesis of osteomyelitis and disease progression. It has been shown that the pathogenic bacteria Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are capable of inducing differentiated production of cytokines. The most attention has been attracted to E. faecium, which exhibits multidrug resistance to a wide range of antibiotics. The proportion of infections mediated by S. epidermidis and S. Saprophyticus is, on average, about 25% of cases. The proportion of representatives of gram-negative flora Escherichia, Klebsiella, Enterobacter, Citrobacter, Proteus, Providencia, Serratia reaches 23% of cases. Pathogenic nosocomial strains of P. aeruginosa are also involved in the formation of chronic inflammation in osteomyelitis. According to the results of published studies, more than a third of cases of chronic osteomyelitis are mediated by microbial associations, which are dominated by S. aureus, S. epidermidis and, less commonly, E. faecalis. Conclusions. The use of PCR analysis to identify the causative agents of osteomyelitis and gene amplification using specific primers has a huge advantage over routine microbiological tests, being an informative method for studying the pathogenicity factors of the main pathogens. The high importance of molecular genetic methods in the study of the etiopathogenesis of osteomyelitis of the jaws requires their widespread use in the clinic of surgical dentistry and maxillofacial surgery to successfully solve complex problems in the rehabilitation of patients with this disease.
Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans , a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S . mutans , we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S . mutans . Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5’-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S . mutans , along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.
Solventogenic Clostridium species are important for establishing sustainable industrial bioproduction of fuels and important chemicals. The inherent versatility of these species in substrate utilization and the range of solvents produced during acetone butanol-ethanol (ABE) fermentation make solventogenic Clostridium an attractive choice for biotechnological applications such as production of fuels and chemicals. The functional qualities of these microbes have thus been identified to be due to complex regulatory networks that play essential roles in modulating the metabolism of this group of bacteria. Genomes of solventogenic Clostridium species have relatively greater prevalence of genes that are intricately controlled by various regulatory molecules than most other species. Consequently, the use of genetic or metabolic engineering strategies that do not consider the underlying regulatory mechanisms will not be effective. Several regulatory factors involved in substrate uptake/utilization, sporulation, solvent production, and stress responses (Carbon Catabolite Protein A, Spo0A, AbrB, Rex, CsrA) have been identified and characterized. In this review, the focus is on newly identified regulatory factors in solventogenic Clostridium species, the interaction of these factors with previously identified molecules, and potential implications on substrate utilization, solvent production, and resistance/tolerance to lignocellulose-derived microbial inhibitory compounds.
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.
A plethora of gene regulatory mechanisms with eccentric attributes in Deinoccocus radiodurans confer it to possess a distinctive ability to survive under ionizing radiation. Among the many regulatory processes, small RNA (sRNA)-mediated regulation of gene expression is prevalent in bacteria but barely investigated in D. radiodurans. In the current study, we identified a novel sRNA, DrsS, through RNA-seq analysis in D. radiodurans cells while exposed to ionizing radiation. Initial sequence analysis for promoter identification revealed that drsS is potentially co-transcribed with sodA and dr_1280 from a single operon. Elimination of the drsS allele in D. radiodurans chromosome resulted in an impaired growth phenotype under γ-radiation. DrsS has also been found to be upregulated under oxidative and genotoxic stresses. Deletion of the drsS gene resulted in the depletion of intracellular concentration of both Mn²⁺ and Fe²⁺ by ~70% and 40%, respectively, with a concomitant increase in carbonylation of intracellular protein. Complementation of drsS gene in ΔdrsS cells helped revert its intracellular Mn²⁺ and Fe²⁺ concentration and alleviated carbonylation of intracellular proteins. Cells with deleted drsS gene exhibited higher sensitivity to oxidative stress than wild-type cells. Extrachromosomally expressed drsS in ΔdrsS cells retrieved its oxidative stress resistance properties by catalase-mediated detoxification of reactive oxygen species (ROS). In vitro binding assays indicated that DsrS directly interacts with the coding region of the katA transcript, thus possibly protecting it from cellular endonucleases in vivo. This study identified a novel small RNA DrsS and investigated its function under oxidative stress in D. radiodurans.
IMPORTANCE
Deinococcus radiodurans possesses an idiosyncratic quality to survive under extreme ionizing radiation and, thus, has evolved with diverse mechanisms which promote the mending of intracellular damages caused by ionizing radiation. As sRNAs play a pivotal role in modulating gene expression to adapt to altered conditions and have been delineated to participate in almost all physiological processes, understanding the regulatory mechanism of sRNAs will unearth many pathways that lead to radioresistance in D. radiodurans. In that direction, DrsS has been identified to be a γ-radiation-induced sRNA, which is also induced by oxidative and genotoxic stresses. DrsS appeared to activate catalase under oxidative stress and detoxify intracellular ROS. This sRNA has also been shown to balance intracellular Mn(II) and Fe concentrations protecting intracellular proteins from carbonylation. This novel mechanism of DrsS identified in D. radiodurans adds substantially to our knowledge of how this bacterium exploits sRNA for its survival under stresses.
Bacterial biofilm has brought a lot of intractable problems in food and biomedicine areas. Conventional biofilm control mainly focuses on inactivation and removal of biofilm. However, with robust construction and enhanced resistance, the established biofilm is extremely difficult to eradicate. According to the mechanism of biofilm development, biofilm formation can be modulated by intervening in the key factors and regulatory systems. Therefore, regulation of biofilm formation has been proposed as an alternative way for effective biofilm control. This review aims to provide insights into the regulation of biofilm formation in food and biomedicine. The underlying mechanisms for early-stage biofilm establishment are summarized based on the key factors and correlated regulatory networks. Recent developments and applications of novel regulatory strategies such as anti/pro-biofilm agents, nanomaterials, functionalized surface materials and physical strategies are also discussed. The current review indicates that these innovative methods have contributed to effective biofilm control in a smart, safe and eco-friendly way. However, standard methodology for regulating biofilm formation in practical use is still missing. As biofilm formation in real-world systems could be far more complicated, further studies and interdisciplinary collaboration are still needed for simulation and experiments in the industry and other open systems.
Introduction. Salmonella Typhimurium (STM) is a food-borne Gram-negative bacterium, which can infect humans and a wide range of livestock and poultry, causing a variety of diseases such as septicaemia, enteritis and abortion.
Hypothesis/Gap Statement. We will decipher the impacts of sRNA STnc1280 on STM virulence and provide a theoretical basis to reveal the regulatory role and molecular mechanism of STnc1280 .
Aim. The main objective of this study was to clarify whether sRNA STnc1280 exerts regulatory roles on STM pathogenicity.
Methodology. The STnc1280 gene was amplified and its molecular characteristics were analysed in this study. Then, STnc1280 gene deletion strain (STM- ΔSTnc1280 ) and the complementary strain ( ΔSTnc1280/STnc1280 ) were constructed by λ-Red homologous recombination method, respectively, to analyse of adhesion and invasive ability and pathogenicity of different strains. Subsequently, the potential target gene regulated by STnc1280 was predicted using target RNA2 software, followed by the verification of the interaction between STnc1280 and target mRNA using the dual plasmid reporter system (DPRS). Furthermore, the mRNA and protein level of target gene was determined using qRT-PCR and Western blot, respectively.
Results. The results revealed that the cell adhesion and invasive ability and pathogenicity of STM- ΔSTnc1280 were significantly reduced compared to STM-SL1344 strain, indicating that the deficiency of STnc1280 gene significantly influenced STM pathogenicity. The DPRS results showed that STnc1280 can interact with the mRNA of target gene gldA , thus suppressing the expression of lacZ gene. Furthermore, the level of gldA mRNA was not influenced in STM- ΔSTnc1280 , but the expression of GldA protein decreased significantly.
Conclusion. Combining the bioinformatic analysis, these findings suggested that STnc1280 may bind to the SD sequence of gldA mRNA, hindering the binding of ribosomes to gldA mRNA, thereby inhibiting the expression of GldA protein to modulate the virulence of STM.
Molecular dynamics simulations have proved extremely useful in investigating the functioning of proteins with atomic-scale resolution. Many applications to the study of RNA also exist, and their number increases by the day. However, implementing MD simulations for RNA molecules in solution faces challenges that the MD practitioner must be aware of for the appropriate use of this tool. In this chapter, we present the fundamentals of MD simulations, in general, and the peculiarities of RNA simulations, in particular. We discuss the strengths and limitations of the technique and provide examples of its application to elucidate small RNA’s performance.
Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at—or migration along—predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.
Small non-coding RNAs (sRNAs) play vital roles in gene expression regulation and RNA interference. To comprehend their molecular mechanisms and develop therapeutic approaches, determining the accurate three-dimensional structure of sRNAs is crucial. Although nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for structural biology, obtaining high-resolution structures of sRNAs using NMR data alone can be challenging. In such cases, structural modeling can provide additional details about RNA structures. In this context, we present a protocol for the structural modeling of sRNA using the SimRNA method based on sparse NMR constraints. To demonstrate the efficacy of our method, we provide selected examples of NMR spectra and RNA structures, specifically for the second stem-loop of DsrA sRNA.
Our review highlights strategies for engineering electron transfer between EAMs and electrodes and discusses the challenges and prospects of these strategies in facilitating electrical energy generation and chemicals synthesis.
Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to functionally characterise environmental adaptation, antibiotic resistance and pathogenicity of this organism and their genetic regulators to inform intervention strategies. Critical to rapid adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners in A. baumannii , we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. We found that 40 sRNA candidates were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii and that sRNAs act as direct regulators of mRNA molecules through antisense base-pairing. In-depth characterisation uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including that of the outer membrane protein CarO and the siderophore receptor BfnH. We show that Aar initiates base-pairing with these mRNA molecules using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in response to multiple stress stimuli suggesting a role in fine-tuning translation of the Aar-target molecules in A. baumannii under hostile conditions. Together, our study provides mechanistic insights into sRNA-mediated gene expression control in A. baumannii and represents a valuable resource for future RNA-centric research endeavours in this ESKAPE pathogen.
The virulence genes of bacterial pathogens are rarely expressed continuously. Instead, their expression is usually confined to times and places when this will be advantageous to the pathogen and avoids the wasteful production of physiologically expensive structures and effectors. Bacteria achieve virulence gene control at every level of gene expression, and they exhibit an impressive capacity for coordinating the expression of virulence genes with genes that perform housekeeping functions. It has become clear that virulence genes in many pathogens have been acquired by horizontal gene transfer, a process in which foreign DNA is taken up by a bacterium and integrated physically into its ancestral gene complement. Physical genetic integration is accompanied by an enmeshing of the new genes in the gene control networks that already exist in the bacterium. These networks are governed by regulatory factors that may be proteins, small RNAs, variable DNA topology, all acting in combination. The imported gene clusters often include regulatory genes too, and these must now work alongside those expressed by the ancestral genome. Regulators operate within hierarchies whose architecture is subject to change, as the circumstances of the bacterium demand. The dynamic nature of the regulatory networks and hierarchies is guided by information coming from the external and internal environments of the bacterial cell. The result is an integrated living machine that is sensitive to its surroundings and can exploit these by the timely expression of virulence determinants. These principles will be illustrated in this chapter using three well-studied examples of bacterial virulence control systems: those of Salmonella enterica serovar typhimurium, Vibrio cholerae, and Shigella flexneri.
Glucose is an important carbon source for microbial growth, and its content in infertile soils is essential for the growth of bacteria. Since the mechanism of oligotrophic bacterium adaptation in barren soils is unclear, this research employed RNA-seq technology to examine the impact of glucose concentration on the oligotrophic bacterium B. subtilis BS-G1 in soil affected by desertification. A global transcriptome analysis (RNA-Seq) revealed that the significantly differentially expressed genes (DEGs) histidine metabolism, glutamate synthesis, the HIF-1 signaling pathway, sporulation, and the TCA cycle pathway of B. subtilis BS-G1 were significantly enriched with a 0.015 g/L glucose concentration (L group), compared to a 10 g/L glucose concentration (H group). The DEGs amino acid system, two-component system, metal ion transport, and nitrogen metabolism system of B. subtilis BS-G1 were significantly enriched in the 5 g/L glucose concentration (M group), compared with the H group. In addition, the present study identified the regulation pattern and key genes under a low-glucose environment (7 mRNAs and 16 sRNAs). This study primarily investigates the variances in the regulatory pathways of the oligotrophic B. subtilis BS-G1, which holds substantial importance in comprehending the mechanism underlying the limited sugar tolerance of oligotrophic bacteria.
Small RNAs (sRNAs), a subset of non-coding RNAs, act posttranscriptionally to salvage bacteria from stress, thereby modulating multiple aspects of bacterial growth, physiology, and virulence. Conventional genetic screens do not efficiently detect sRNAs, rendering most sRNAs in E. coli uncharacterized. Characterizing sRNA regulons and their mechanism(s) of action is necessary for understanding the regulatory networks controlling growth, physiology, and virulence. Utilizing the sRNAMap database and the deposited expression datasets on the NCBI Gene Expression Omnibus (GEO), we chose a pool of uncharacterized E. coli sRNAs that are differentially expressed under distinct stress conditions. These sRNAs were then cloned into medium copy-number plasmids. Simultaneously, IntaRNA was used to computationally predict potential targets for these sRNAs. To validate posttranscriptional regulation of these sRNA targets, we constructed target lacZ translational fusions using lambda-Red recombineering. We confirmed posttranscriptional regulation by measuring changes in the β-galactosidase activity of these fusions upon ectopic expression of the sRNAs. Successful completion of this project will help elucidate the individual targetomes of these novel sRNAs. Ultimately, this will aid our understanding of the physiological condition(s) that trigger the expression of these sRNAs.
Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose rationally designed new small RNAs (sRNAs) that dynamically modulate gene expression of genetic circuits with a broad range (high, medium, and low) of repression. We designed multiple multilayer genetic circuits in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein. The sRNAs target TFs instead of a reporter gene, and harnessing the intrinsic RNA-interference pathway in E. coli allowed for a wide range of expression modulation of the reporter protein, including the most difficult to achieve dynamic switch to an OFF state. The synthetic sRNAs are expressed independently of the circuit(s), thus allowing for repression without modifying the circuit itself. Our work provides a frame for achieving independent modulation of gene expression and dynamic and modular control of the multilayer genetic circuits by only including an independent control circuit expressing synthetic sRNAs, without altering the structure of existing genetic circuits.
Bacterial small RNAs (sRNAs) regulate many important physiological processes in cells, including antibiotic resistance and virulence genes, through base-pairing interactions with mRNAs. Antisense oligonucleotides (ASOs) have great potential as therapeutics against bacterial pathogens by targeting sRNAs such as MicF, which regulates outer membrane protein OmpF expression and limits the permeability of antibiotics. Here we devised a cell-free transcription-translation (TX-TL) assay to identify ASO designs that sufficiently sequester MicF. ASOs were then ordered as peptide nucleic acids conjugated to cell-penetrating peptides (CPP-PNA) to allow for effective delivery into bacteria. Subsequent minimum inhibitory concentration (MIC) assays demonstrated that simultaneously targeting the regions of MicF responsible for sequestering the start codon and the Shine-Dalgarno sequence of ompF with two different CPP-PNAs synergistically reduced the MIC for a set of antibiotics. This investigation offers a TX-TL-based approach to identify novel therapeutic candidates to combat intrinsic sRNA-mediated antibiotic resistance mechanisms.
Neisseria gonorrhoeae is a human-specific pathogen, causing the sexually transmitted disease gonorrhea. Gonococci are adapted at evading the immune response, but although an uncomplicated infection can be easily treated, the increase in antibiotic resistance is the cause of concern. Here we present our latest contributions to N. gonorrhoeae research, elucidating various aspects of the infection and the role of known and novel pathogenicity factors in the interaction of these bacteria with the host.
The mammalian intestinal tract harbors trillions of microorganisms confined within this space by mucosal barriers. Despite these barriers, bacterial components may still be found elsewhere in the body, even in healthy subjects. Bacteria can release small lipid-bound particles, also named bacterial extracellular vesicles (bEV). While bacteria themselves cannot normally penetrate the mucosal defense, bEVs may infiltrate the barrier and disseminate throughout the body. The extremely diverse cargo that bEVs can carry, depending on their parent species, strain, and growth conditions, grant them an equally broad potential to interact with host cells and influence immune functions. Herein, we review the current knowledge of processes underlying the uptake of bEVs by mammalian cells, and their effect on the immune system. Furthermore, we discuss how bEVs could be targeted and manipulated for diverse therapeutic purposes.
Bacillus amyloliquefaciens is an interesting microbe in the food processing and manufacturing industries. Non-coding small RNAs (sRNAs) have been shown to play a crucial role in the physiology and metabolism of bacteria by post-transcriptionally regulating gene expression. This study investigated the function of novel sRNA FenSr3 by constructing fenSr3 deficient strain and complementary strains in B. amyloliquefaciens LPB-18 , which were named LPN-18N and LPB-18P, respectively. The result showed significant differences in fengycin yield between strain LPB -18N and LPB-18P. The production of fengycin was significantly enhanced in B. amyloliquefaciens LPB-18N, compared with that of the strain LPB-18 from 190.908 mg/L to 327.598 mg/L. Moreover, the production of fengycin decreased from 190.464 mg/L to 38.6 mg/L in B . amyloliquefaciens LPB-18P. A comparative transcriptome sequencing was carried out to better understand the complex regulatory mechanism. Transcription analysis revealed that 1037 genes were differentially expressed between B. amyloliquefaciens LPB-18 and B. amyloliquefaciens LPB-18N, including the key regulatory genes in fatty acid, amino acid biosynthesis, and central carbon metabolism, which could provide sufficient quantities of building precursors for fengycin biosynthesis. The biofilm formation and sporulation was also enhanced in the strain LPB-18N, which indicates that FenSr3 could play a vital role in stress resistance and promotes survival in B. amyloliquefaciens . Some sRNAs involved in stress response have been identified in the literature, but their regulatory roles in fengycin production remain unclear. The study will contribute a novel perspective to the regulation mechanism of biosynthesis and the optimization of key metabolites of B. amyloliquefaciens.
The relationship between rates of genomic evolution and organismal adaptation remains uncertain, despite considerable interest. The feasibility of obtaining genome sequences from experimentally evolving populations offers the opportunity to investigate this relationship with new precision. Here we sequence genomes sampled through 40,000 generations from a laboratory population of Escherichia coli. Although adaptation decelerated sharply, genomic evolution was nearly constant for 20,000 generations. Such clock-like regularity is usually viewed as the signature of neutral evolution, but several lines of evidence indicate that almost all of these mutations were beneficial. This same population later evolved an elevated mutation rate and accumulated hundreds of additional mutations dominated by a neutral signature. Thus, the coupling between genomic and adaptive evolution is complex and can be counterintuitive even in a constant environment. In particular, beneficial substitutions were surprisingly uniform over time, whereas neutral substitutions were highly variable.
The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function.
Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.
Sequence-function relations for small RNA (sRNA)-mediated gene silencing were quantified for the sRNA RyhB and some of its mRNA targets in Escherichia coli. Numerous mutants of RyhB and its targets were generated and their in vivo functions characterized at various levels of target and RyhB expression. Although a core complementary region is required for repression by RyhB, variations in the complementary sequences of the core region gave rise to a continuum of repression strengths, correlated exponentially with the computed free energy of RyhB-target duplex formation. Moreover, sequence variations in the linker region known to interact with the RNA chaperone Hfq also gave rise to a continuum of repression strengths, correlated exponentially with the computed energy cost of keeping the linker region open. These results support the applicability of the thermodynamic model in predicting sRNA-mRNA interaction and suggest that sequences at these locations may be used to fine-tune the degree of repression. Surprisingly, a truncated RyhB without the Hfq-binding region is found to repress multiple targets of the wild-type RyhB effectively, both in the presence and absence of Hfq, even though the former is required for the activity of wild-type RyhB itself. These findings challenge the commonly accepted model concerning the function of Hfq in gene silencing-both in providing stability to the sRNAs and in catalyzing the target mRNAs to take on active conformations-and raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences.
The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3' end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3' hydroxyl group. A free 3' hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3' end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.
The bacterial envelope stress response (ESR) is triggered by the accumulation of misfolded outer membrane proteins (OMPs) upon envelope damage or excessive OMP synthesis, and is mediated by the alternative sigma factor, sigmaE. Activation of the GE pathway causes a rapid downregulation of major omp mRNAs, which prevents further build-up of unassembled OMPs and liberates the translocation and folding apparatus under conditions that require envelope remodelling. The factors that facilitate the rapid removal of the unusually stable omp mRNAs in the ESR were previously unknown. We report that in Salmonella the ESR relies upon two highly conserved, sigmaE-controlled small non-coding RNAs, RybB and MicA. By using a transcriptomic approach and kinetic analyses of target mRNA decay in vivo, RybB was identified as the factor that selectively accelerates the decay of multiple major omp mRNAs upon induction of the ESR, while MicA is proposed to facilitate rapid decay of the single ompA mRNA. In unstressed bacterial cells, the two oE-dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of oE.
The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.
A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.
The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.
Second generation sequencing has prompted a number of groups to re-interrogate the transcriptomes of several bacterial and archaeal species. One of the central findings has been the identification of complex networks of small non-coding RNAs that play central roles in transcriptional regulation in all growth conditions and for the pathogen's interaction with and survival within host cells. Legionella pneumophila is a gram-negative facultative intracellular human pathogen with a distinct biphasic lifestyle. One of its primary environmental hosts in the free-living amoeba Acanthamoeba castellanii and its infection by L. pneumophila mimics that seen in human macrophages. Here we present analysis of strand specific sequencing of the transcriptional response of L. pneumophila during exponential and post-exponential broth growth and during the replicative and transmissive phase of infection inside A. castellanii. We extend previous microarray based studies as well as uncovering evidence of a complex regulatory architecture underpinned by numerous non-coding RNAs. Over seventy new non-coding RNAs could be identified; many of them appear to be strain specific and in configurations not previously reported. We discover a family of non-coding RNAs preferentially expressed during infection conditions and identify a second copy of 6S RNA in L. pneumophila. We show that the newly discovered putative 6S RNA as well as a number of other non-coding RNAs show evidence for antisense transcription. The nature and extent of the non-coding RNAs and their expression patterns suggests that these may well play central roles in the regulation of Legionella spp. specific traits and offer clues as to how L. pneumophila adapts to its intracellular niche. The expression profiles outlined in the study have been deposited into Genbank's Gene Expression Omnibus (GEO) database under the series accession GSE27232.
The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular
beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing
between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration
but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate.
A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was
faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete.
A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates
helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but
the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding
interactions.
Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems. Molecular Systems Biology 7: 471; published online 1 March 2011; doi:10.1038/msb.2011.4
Hfq is a key regulator involved in multiple aspects of stress tolerance and virulence of bacteria. There has been an intriguing
question as to how this RNA chaperone achieves two completely opposite functions—annealing and unwinding—for different RNA
substrates. To address this question, we studied the Hfq-mediated interaction of fragments of a non-coding RNA, DsrA, with
its mRNA target rpoS by using single-molecule fluorescence techniques. These experiments permitted us to observe the mechanistic steps of Hfq-mediated
RNA annealing/unwinding at the single-molecule level, for the first time. Our real-time observations reveal that, even if
the ring-shaped Hfq displays multiple binding sites for its interaction with RNA, the regulatory RNA and the mRNA compete
for the same binding site. The competition makes the RNA-Hfq interaction dynamic and, surprisingly, increases the overall
annealing efficiency by properly aligning the two RNAs. We furthermore reveal that when Hfq specifically binds to only one
of the two RNAs, the unwinding process dominates over the annealing process, thus shedding a new light on the substrate selectivity
for annealing or unwinding. Finally, our results demonstrate for the first time that a single Hfq hexamer is sufficient to
facilitate sRNA–mRNA annealing.
The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function
of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the
MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction
with the phosphate on the 5′-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed,
eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the
smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine
sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery
and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the
first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated
regulation of stress adaptation in S. meliloti.
Small RNA (sRNA)-induced mRNA degradation occurs through binding of an sRNA to a target mRNA with the concomitant action of the RNA degradosome, which induces an endoribonuclease E (RNase E)-dependent cleavage and degradation of the targeted mRNA. Because many sRNAs bind at the ribosome-binding site (RBS), it is possible that the resulting translation block is sufficient to promote the rapid degradation of the targeted mRNA. Contrary to this mechanism, we report here that the pairing of the sRNA RyhB to the target mRNA sodB initiates mRNA degradation even in the absence of translation on the mRNA target. Remarkably, even though it pairs at the RBS, the sRNA RyhB induces mRNA cleavage in vivo at a distal site located >350 nucleotides (nt) downstream from the RBS, ruling out local cleavage near the pairing site. Both the RNA chaperone Hfq and the RNA degradosome are required for efficient cleavage at the distal site. Thus, beyond translation initiation block, sRNA-induced mRNA cleavage requires several unexpected steps, many of which are determined by structural features of the target mRNA.
Bacterial small noncoding RNAs have attracted much interest in recent years as posttranscriptional regulators of genes involved
in diverse pathways. Small RNAs (sRNAs) are 50 to 400 nucleotides long and exert their regulatory function by directly base
pairing with mRNA targets to alter their stability and/or affect their translation. This base pairing is achieved through
a region of about 10 to 25 nucleotides, which may be located at various positions along different sRNAs. By compiling a data
set of experimentally determined target-binding regions of sRNAs and systematically analyzing their properties, we reveal
that they are both more evolutionarily conserved and more accessible than random regions. We demonstrate the use of these
properties for computational identification of sRNA target-binding regions with high specificity and sensitivity. Our results
show that these predicted regions are likely to base pair with known targets of an sRNA, suggesting that pointing out these
regions in a specific sRNA can help in searching for its targets.
Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium,
together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional
Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full
virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form
trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These
results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and
virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might
play additional roles besides acting as an RNA chaperone.
Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.
There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.
MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes.
While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack
of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA
silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments,
we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated
into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality
of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design
of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond
to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help
advance applications in biological research and applied medicine.
MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role
of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control
of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5′ phosphorylation
state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves
a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage
of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the
eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.
Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K(d) values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1- 2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are ≈1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the "strong binding-high turnover" paradox and permits efficient use of the Hfq pool.
The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5' located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5' end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5' domain is sufficient to guide target mRNA degradation and maintain σ(E)-dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3' adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5' domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.
In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.
It is well known that 6S RNA, a global regulatory noncoding RNA that modulates gene expression in response to the cellular stresses in Escherichia coli, is generated by processing from primary ssrS (6S RNA) transcripts derived from two different promoters. The 5' processing of 6S RNA from primary transcripts has been well studied; however, it remains unclear how the 3'-end of this RNA is generated although previous studies have suggested that exoribonucleolytic trimming is necessary for 3' processing. Here, we describe several Rho-dependent termination sites located ∼90 bases downstream of the mature 3'-end of 6S RNA. Our data suggest that the 3'-end of 6S RNA is generated via exoribonucleolytic trimming, rather than endoribonucleolytic cleavage, following the transcription termination events. The termination sites identified in this study are within the open reading frame of the downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase) gene, a part of the highly conserved bacterial operon ssrS-ygfA, which is up-regulated during the biofilm formation. Our findings reveal that ygfA expression, which also aids the formation of multidrug-tolerant persister cells, could be regulated by Rho-dependent termination activity in the cell.
Genome-wide studies of bacterial gene expression are shifting from microarray technology to second generation sequencing platforms. RNA-seq has a number of advantages over hybridization-based techniques, such as annotation-independent detection of transcription, improved sensitivity and increased dynamic range. Early studies have uncovered a wealth of novel coding sequences and non-coding RNA, and are revealing a transcriptional landscape that increasingly mirrors that of eukaryotes. Already basic RNA-seq protocols have been improved and adapted to looking at particular aspects of RNA biology, often with an emphasis on non-coding RNAs, and further refinements to current techniques will improve our understanding of gene expression, and genome content, in the future.
RNA molecules as sensors and switchesSolving gene control mysteriesCommon riboswitch architecturesBiochemical and genetic validation of riboswitch candidatesBioinformatics approaches to riboswitch discoveryMany bacterial riboswitch classes remain to be discoveredStructural and mechanistic complexity of riboswitchesConclusion
References
An expanding number of metabolite-binding riboswitch classes are being discovered in the noncoding portions of bacterial genomes. Findings over the last decade indicate that bacteria commonly use these RNA genetic elements as regulators of metabolic pathways and as mediators of changes in cell physiology. Some riboswitches are surprisingly complex, and they rival protein factors in their structural and functional sophistication. Each new riboswitch discovery expands our knowledge of the biochemical capabilities of RNA, and some give rise to new questions that require additional study to be addressed. Some of the greatest prospects for riboswitch research and some of the more interesting mysteries are discussed in this review.
The importance of small RNA (sRNA) regulators has been recognized across all domains of life. In bacteria, sRNAs typically control the expression of virulence and stress response genes via antisense base pairing with mRNA targets. Originally dubbed "non-coding RNAs," a number of bacterial antisense sRNAs have been found to encode functional proteins. Although very few of these dual-function sRNAs have been characterized, they have been found in both gram-negative and gram-positive organisms. Among the few known examples, the functions and mechanisms of regulation by dual-function sRNAs are variable. Some dual-function sRNAs depend on the RNA chaperone Hfq for base pairing-dependent regulation (riboregulation); this feature appears so far exclusive to gram-negative bacterial sRNAs. Other variations can be found in the spatial organization of the coding region with respect to the riboregulation determinants. How the functions of encoded proteins relate to riboregulation is for the most part not understood. However, in one case it appears that there is physiological redundancy between protein and riboregulation functions. This mini-review focuses on the two best-studied bacterial dual-function sRNAs: RNAIII from Staphylococcus aureus and SgrS from Escherichia coli and includes a discussion of what is known about the structure, function and physiological roles of these sRNAs as well as what questions remain outstanding.
Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.
GcvB is one of the most highly conserved Hfq-associated small RNAs in Gram-negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB-mediated regulation in Salmonella, we combined a genome-wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild-type versus mutant sRNA variants revealed that GcvB governs a large post-transcriptional regulon, impacting ~1% of all Salmonella genes via its conserved G/U-rich domain R1. Complementary predictions of C/A-rich binding sites in mRNAs and gfp reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base-pairing. This novel ability of GcvB is focused upon the one target that could feedback-regulate the glycine-responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.
Under conditions of nutrient deprivation or stress, or as cells enter stationary phase, Escherichia coli and related bacteria increase the accumulation of RpoS, a specialized sigma factor. RpoS-dependent gene expression leads to general stress resistance of cells. During rapid growth, RpoS translation is inhibited and any RpoS protein that is synthesized is rapidly degraded. The complex transition from exponential growth to stationary phase has been partially dissected by analyzing the induction of RpoS after specific stress treatments. Different stress conditions lead to induction of specific sRNAs that stimulate RpoS translation or to induction of small-protein antiadaptors that stabilize the protein. Recent progress has led to a better, but still far from complete, understanding of how stresses lead to RpoS induction and what RpoS-dependent genes help the cell deal with the stress.
Gene expression in eukaryotes is subject to extensive regulation at posttranscriptional levels. One of the most important sites of control involves mRNA 3' untranslated regions (3'UTRs), which are recognized by RNA-binding proteins (RBPs) and microRNAs (miRNAs). These factors greatly influence translational efficiency and stability of target mRNAs and often also determine their cellular localization. HuR, a ubiquitously expressed member of the ELAV family of RBPs, has been implicated in regulation of stability and translation of over one hundred mRNAs in mammalian cells. Recent data indicate that some of the effects of HuR can be explained by its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of miRNAs interacting with the 3'UTR and redirect the repressed mRNA to polysomes for active translation. However, HuR can also synergize with miRNAs. The finding that HuR is able to disengage miRNAs from the repressed mRNA, or render them inactive, provides evidence that miRNA regulation is much more dynamic then originally anticipated. In this chapter we review properties of HuR and describe examples of the cross-talk between the protein and miRNAs, with emphasis on response of the regulation to cellular stress.
Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.
In Escherichia coli, many small noncoding regulatory RNAs (sRNAs) post-transcriptionally regulate gene expression by base-pairing to mRNAs in a process that is mediated by the RNA chaperone Hfq. Binding of the sRNA to the mRNA can lead to increased or decreased mRNA stability and/or translation. It is not known if proteins other than Hfq are necessary for this process. In order to identify additional genes required for the post-transcriptional regulation of gene expression by Hfq-dependent sRNAs, we developed a novel combined genetic selection and screen for mutants defective in sRNA regulation. In our combined genetic selection and screen, we isolated hfq mutants and mutants in pnp, encoding polynucleotide phosphorylase (PNPase). We show that loss-of-function mutations in pnp result in a decreased stability of several sRNAs including RyhB, SgrS, and CyaR and also decrease both the negative and positive regulation by sRNAs. The defect in stability of CyaR and in negative and positive regulation are suppressed by deletion mutations in RNase E. Altogether, our results suggest that the lack of sRNA-mediated regulation in the absence of an active form of PNPase is due to the rapid turnover of sRNA resulting from an increase in RNase E activity and/or an increase in access of other ribonucleases to sRNAs.
The binding of nine noncoding regulatory RNAs (sRNAs) to the E. coli Hfq protein was compared using a high-throughput double filter retention assay. Despite the fact that these sRNAs have different lengths, sequences and secondary structures their Hfq binding affinities were surprisingly uniform. The analysis of sRNAs binding to Hfq mutants showed that the proximal face of Hfq, known as the binding site for DsrA RNA, is a universal sRNA binding site. Moreover, all sRNAs bound Hfq with similar association rates limited only by the rate of diffusion, while the rates of dissociation, measured in the dilution experiments, were uniformly slow. Despite that, the data showed that there was a hierarchy of sRNAs in regard to their performance in competition for access to Hfq and in their ability to facilitate the dissociation of other sRNAs from Hfq. The sRNAs also differed in their salt dependence of binding to this protein. Overall, the results suggest that despite the uniform binding of different sRNAs to the same site on Hfq their exchange on this protein is dependent on the identities of the competing sRNAs.
Synthetic biology aims to make the engineering of biology faster and more predictable. In contrast, systems biology focuses on the interaction of myriad components and how these give rise to the dynamic and complex behavior of biological systems. Here, we examine the synergies between these two fields.
Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.
Bacteria cycle between periods when they perform individual behaviors and periods when they perform group behaviors. These transitions are controlled by a cell-cell communication process called quorum sensing, in which extracellular signal molecules, called autoinducers (AIs), are released, accumulate, and are synchronously detected by a group of bacteria. AI detection results in community-wide changes in gene expression, enabling bacteria to collectively execute behaviors such as bioluminescence, biofilm formation, and virulence factor production. In this study, we show that the transcription factor AphA is a master regulator of quorum sensing that operates at low cell density (LCD) in Vibrio harveyi and Vibrio cholerae. In contrast, LuxR (V. harveyi)/HapR (V. cholerae) is the master regulator that operates at high cell density (HCD). At LCD, redundant small noncoding RNAs (sRNAs) activate production of AphA, and AphA and the sRNAs repress production of LuxR/HapR. Conversely, at HCD, LuxR/HapR represses aphA. This network architecture ensures maximal AphA production at LCD and maximal LuxR/HapR production at HCD. Microarray analyses reveal that 300 genes are regulated by AphA at LCD in V. harveyi, a subset of which is also controlled by LuxR. We propose that reciprocal gradients of AphA and LuxR/HapR establish the quorum-sensing LCD and HCD gene expression patterns, respectively.
Chemically inducible RNA interference (RNAi) enables temporal and/or spatial control of virtually any gene, making it useful for study of gene functions, discovery of potential drug targets, and gene therapy applications. Here we describe a new inducible RNAi platform in which orthogonal chemically modified oligonucleotides are used to trigger silencing of two genes in a combinatorial manner. We developed a modular RNA architecture consisting of an oligonucleotide sensor stem-loop and an RNAi effector domain that is designed to undergo a structural shift upon addition of an oligonucleotide inducer. The induced structural change allows the RNA to be processed by the RNAi machinery, ultimately resulting in gene silencing of the target encoded by the RNAi effector module. Combinatorial regulation of multiple genes should accelerate studies of complex gene-gene interactions and screening of new drug targets.
Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base-pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse nonpreferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multioutput feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base-pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression.
An RNA chaperon Hfq along with Hfq-binding sRNAs stably binds to RNase E in Escherichia coli. The role of the Hfq-RNase E interaction is to recruit RNase E to target mRNAs of sRNAs resulting in the rapid degradation of the mRNA-sRNA hybrid. The C-terminal scaffold region of RNase E is responsible for the interaction with Hfq. Here, we demonstrate that the scaffold region can be deleted up to residue 750 without losing the ability to cause the rapid degradation of target mRNAs mediated by Hfq/sRNAs. The truncated RNase E750 can still bind to Hfq although the truncation significantly reduces the Hfq-binding ability. We conclude that the subregion between 711 and 750 is sufficient for the functional interaction with Hfq to support the rapid degradation of ptsG mRNA although additional subregions within the scaffold are also involved in Hfq binding. Deletion of the 702-750 region greatly impairs the ability of RNase E to cause the degradation of ptsG mRNA. In addition, a polypeptide corresponding to the scaffold region binds to Hfq without the help of RNA. Finally, we demonstrate that overexpression of RhlB partially inhibits the Hfq binding to RNase E and the rapid degradation of ptsG mRNA.
Small RNAs (sRNAs) regulate diverse pathways, including stress responses, virulence, and metabolism in Escherichia coli. At the center of this large sRNA regulatory network is the Hfq protein. Hfq mediates the binding of sRNAs to their target mRNAs; without Hfq, most sRNAs cannot efficiently regulate target mRNA expression. Here, we show in vivo that Hfq can be a limiting factor for sRNA activity and that it can be easily depleted, causing disruption of the sRNA network. Depletion of the available Hfq can occur when sRNAs and target mRNAs are transcribed at high levels without their partners, resulting in the sequestration of Hfq into sRNA-Hfq and target mRNA-Hfq complexes. This can be avoided by coordinating the transcription of sRNAs with their target mRNAs so that they are turned on and off together to maximize duplex formation and minimize Hfq sequestration. Therefore, the limited availability of Hfq results in a highly interdependent sRNA network, wherein the activity of each sRNA depends on the activity of the other sRNAs and target mRNAs in the network.
The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.
The adjacent gadX and gadW genes encode transcription regulators that are part of a complex regulatory circuit controlling the Escherichia coli response to acid stress. We previously showed that the small RNA GadY positively regulates gadX mRNA levels. The gadY gene is located directly downstream of the gadX coding sequence on the opposite strand of the chromosome. We now report that gadX is transcribed in an operon with gadW, although this full-length mRNA does not accumulate. Base pairing of the GadY small RNA with the intergenic region of the gadX-gadW mRNA results in directed processing events within the region of complementarity. The resulting two halves of the cleaved mRNA accumulate to much higher levels than the unprocessed mRNA. We examined the ribonucleases required for this processing, and found that multiple enzymes are involved in the GadY-directed cleavage including the double-strand RNA-specific endoribonuclease RNase III.
Small RNA molecules play key regulatory roles in many bacterial species. However, little mechanistic data exists for the action of small regulatory RNAs in the human pathogen group A Streptococcus (GAS). Here, we analysed the relationship between a putative GAS sRNA and production of the secreted virulence factor streptokinase (SKA). SKA promotes GAS dissemination by activating conversion of host plasminogen into the fibrin-degrading protease plasmin. Homologues of the putative sRNA-encoding gene fibronectin/fibrinogen-binding/haemolytic-activity/streptokinase-regulator-X (fasX) were identified in four different pyogenic streptococcal species. However, despite 79% fasX nucleotide identity, a fasX allele from the animal pathogen Streptococcus zooepidemicus failed to complement a GAS fasX mutant. Using a series of precisely constructed fasX alleles we discovered that FasX is a bona-fide sRNA that post-transcriptionally regulates SKA production in GAS. By base-pairing to the 5' end of ska mRNA, FasX enhances ska transcript stability, resulting in a ∼10-fold increase in SKA activity. Our data provide new insights into the mechanisms used by small regulatory RNAs to activate target mRNAs, and enhances our understanding of the regulation of a key GAS virulence factor.
Prokaryotic regulatory small RNAs act by a conserved mechanism and yet display a stunning structural variability. In the present study, we used mutational analysis to dissect the functional anatomy of RybB, a σ(E)-dependent sRNA that regulates the synthesis of major porins in Escherichia coli and Salmonella. Mutations in the chromosomal rybB locus that altered the expression of an ompC-lac fusion were identified. Some of the mutations cluster within a seven-nucleotide segment at the 5' end of the sRNA and affect its ability to pair with a sequence 40 nucleotides upstream from ompC translation start site. Other mutations map near the 3' end of RybB, destabilizing the sRNA or altering its binding to Hfq. The 5' end of RybB is also involved in ompD regulation. In this case, the sRNA can choose between two mutually exclusive pairing sites within the translated portion of the mRNA. Some of the RybB 5' end mutations affect the choice between the two sites, resulting in regulatory responses that diverge from those observed in ompC. Further analysis of RybB target specificity identified chiP (ybfM), a gene encoding an inducible chitoporin, as an additional member of the RybB regulon. Altogether, our results indicate that an heptameric 'seed' sequence is sufficient to confer susceptibility to RybB regulation.
MicroRNA (miRNA) modules are built in genetic networks as a complex regulatory layer directing post-transcriptional gene regulation. miRNAs coordinate a broad spectra of gene expression programs mainly through modulation of mRNA metabolism. Perturbations of miRNA networks are linked to a wide variety of pathological processes, including cardiovascular diseases and cancer. While the mechanisms regulating miRNA biogenesis were previously poorly understood, recent findings have shed light on the regulatory mechanisms of miRNAs themselves, especially their biogenesis. Multiple steps of miRNA maturation could potentially provide a variety of regulatory options to generate mature miRNAs differentially and produce gradation in miRNA processing efficiency. Several studies have demonstrated that miRNA maturation pathways crosstalk with intracellular signalling molecules, including p53, Smad proteins and estrogen receptor. Other lines of evidence have demonstrated the involvement of multiple RNA binding proteins in biased processing of different miRNA species. This review summarizes accumulating evidence for the emerging complexity and dynamics of regulated miRNA processing. These findings will lead to better understanding of miRNA dynamics in various pathogenetic pathways and provide the molecular basis for diagnostic and therapeutic strategies based on small RNA biology.
6S RNA is a small, non-coding RNA that interacts directly with σ(70)-RNA polymerase and regulates transcription at many σ(70)-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ(70)-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σ(S) activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions.
Cancer cells are characterized by genetic mutations that deregulate cell proliferation and suppress cell death. To arrest the uncontrolled replication of malignant cells, conventional chemotherapies systemically disrupt cell division, causing diverse and often severe side effects as a result of collateral damage to normal cells. Seeking to address this shortcoming, we pursue therapeutic regulation that is conditional, activating selectively in cancer cells. This functionality is achieved using small conditional RNAs that interact and change conformation to mechanically transduce between detection of a cancer mutation and activation of a therapeutic pathway. Here, we describe small conditional RNAs that undergo hybridization chain reactions (HCR) to induce cell death via an innate immune response if and only if a cognate mRNA cancer marker is detected within a cell. The sequences of the small conditional RNAs can be designed to accept different mRNA markers as inputs to HCR transduction, providing a programmable framework for selective killing of diverse cancer cells. In cultured human cancer cells (glioblastoma, prostate carcinoma, Ewing's sarcoma), HCR transduction mediates cell death with striking efficacy and selectivity, yielding a 20- to 100-fold reduction in population for cells containing a cognate marker, and no measurable reduction otherwise. Our results indicate that programmable mechanical transduction with small conditional RNAs represents a fundamental principle for exploring therapeutic conditional regulation in living cells.