RESUMEN

Understanding how cells sense temperature is a fundamental question in biology and is pivotal for the evolution of life. In numerous organisms, temperature is not only sensed but also generated due to cellular processes. Consequently, the mechanisms governing temperature sensation in various organisms have been experimentally elucidated. Extending upon others' proposals and demonstration of protein- and nucleic acid-based thermosensors, and utilizing a colonial India 'punkah-wallahs' analogy, I present my rationale for the necessity of temperature sensing in every organelle in a cell. Finally, I propose temperature-sensing riboceptors (ribonucleic acid receptors) to integrate all the RNA molecules (mRNA, non-coding RNA, and so forth) capable of sensing temperature and triggering a signaling event, which I call as thermocrine signaling. This approach could enable the identification of riboceptors in every cell of almost every organism, not only for temperature but also for other classes of ligands, including gaseous solutes, and water.

Asunto(s)

RESUMEN

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200 nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

Asunto(s)

RESUMEN

RNA thermometers are highly structured noncoding RNAs located in the 5'-untranslated regions (UTRs) of genes that regulate expression by undergoing conformational changes in response to temperature. The discovery of RNA thermometers through bioinformatics is difficult because there is little sequence conservation among their structural elements. Thus, the abundance of these thermosensitive regulatory structures remains unclear. Herein, to advance the discovery and validation of RNA thermometers, we developed Robo-Therm, a pipeline that combines an adaptive and user-friendly in silico motif search with a well-established reporter system. Through our application of Robo-Therm, we discovered two novel RNA thermometers in bacterial and bacteriophage genomes found in the human gut. One of these thermometers is present in the 5'-UTR of a gene that codes for σ 70 RNA polymerase subunit in the bacteria Mediterraneibacter gnavus and Bacteroides pectinophilus, and in the bacteriophage Caudoviricetes, which infects B. pectinophilus The other thermometer is in the 5'-UTR of a tetracycline resistance gene (tetR) in the intestinal bacteria Escherichia coli and Shigella flexneri Our Robo-Therm pipeline can be applied to discover multiple RNA thermometers across various genomes.

Asunto(s)

RESUMEN

Despite possessing attractive features such as autotrophic growth on minimal media, industrial applications of cyanobacteria are hindered by a lack of genetic manipulative tools. There are two important features that are important for an effective manipulation: a vector which can carry the gene, and an induction system activated through external stimuli, giving us control over the expression. In this study, we describe the construction of an improved RSF1010-based vector as well as a temperature-inducible RNA thermometer. RSF1010 is a well-studied incompatibility group Q (IncQ) vector, capable of replication in most Gram negative, and some Gram positive bacteria. Our designed vector, named pSM201v, can be used as an expression vector in some Gram positive and a wide range of Gram negative bacteria including cyanobacteria. An induction system activated via physical external stimuli such as temperature, allows precise control of overexpression. pSM201v addresses several drawbacks of the RSF1010 plasmid; it has a reduced backbone size of 5189 bp compared to 8684 bp of the original plasmid, which provides more space for cloning and transfer of cargo DNA into the host organism. The mobilization function, required for plasmid transfer into several cyanobacterial strains, is reduced to a 99 bp region, as a result that mobilization of this plasmid is no longer linked to the plasmid replication. The RNA thermometer, named DTT1, is based on a RNA hairpin strategy that prevents expression of downstream genes at temperatures below 30 °C. Such RNA elements are expected to find applications in biotechnology to economically control gene expression in a scalable manner.

RESUMEN

The type III secretion system (T3SS) is indispensable for successful host cell infection by many Gram-negative pathogens. The molecular syringe delivers effector proteins that suppress the host immune response. Synthesis of T3SS components in Yersinia pseudotuberculosis relies on host body temperature, which induces the RNA thermometer (RNAT)-controlled translation of lcrF coding for a virulence master regulator that activates transcription of the T3SS regulon. The assembly of the secretion machinery follows a strict coordinated succession referred to as outside-in assembly, in which the membrane ring complex and the export apparatus represent the nucleation points. Two components essential for the initial assembly are YscJ and YscT. While YscJ connects the membrane ring complex with the export apparatus in the inner membrane, YscT is required for a functional export apparatus. Previous transcriptome-wide RNA structuromics data suggested the presence of unique intercistronic RNATs upstream of yscJ and yscT. Here, we show by reporter gene fusions that both upstream regions confer translational control. Moreover, we demonstrate the temperature-induced opening of the Shine-Dalgarno region, which facilitates ribosome binding, by in vitro structure probing and toeprinting methods. Rationally designed thermostable RNAT variants of the yscJ and yscT thermometers confirmed their physiological relevance with respect to T3SS assembly and host infection. Since we have shown in a recent study that YopN, the gatekeeper of type III secretion, also is under RNAT control, it appears that the synthesis, assembly and functionality of the Yersinia T3S machinery is coordinated by RNA-based temperature sensors at multiple levels.

Asunto(s)

RESUMEN

Precise regulation of gene expression is crucial for living cells to adapt for survival in diverse environmental conditions. Among the common cellular regulatory mechanisms, RNA-based regulators play a key role in all domains of life. Discovery of regulatory RNAs have made a paradigm shift in molecular biology as many regulatory functions of RNA have been identified beyond its canonical roles as messenger, ribosomal and transfer RNA. In the complex regulatory RNA network, riboswitches, small RNAs, and RNA thermometers can be identified as some of the key players. Herein, we review the discovery, mechanism, and potential therapeutic use of these classes of regulatory RNAs mainly found in bacteria. Being highly adaptive organisms that inhabit a broad range of ecological niches, bacteria have adopted tight and rapid-responding gene regulation mechanisms. This review aims to highlight how bacteria utilize versatile RNA structures and sequences to build a sophisticated gene regulation network.

RESUMEN

Survival of microorganisms depends to a large extent on environmental conditions and the occupied host. By adopting specific strategies, microorganisms can thrive in the surrounding environment and, at the same time, preserve their viability. Evading the host defenses requires several mechanisms compatible with the host survival which include the production of RNA thermometers to regulate the expression of genes responsible for heat or cold shock as well as of those involved in virulence. Microorganisms have developed a variety of molecules in response to the environmental changes in temperature and even more specifically to the host they invade. Among all, RNA-based regulatory mechanisms are the most common ones, highlighting the importance of such molecules in gene expression control and novel drug development by suitable structure-based alterations. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Asunto(s)

RESUMEN

The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

Asunto(s)

RESUMEN

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

Asunto(s)

RESUMEN

The microbial production of rhamnolipids has been in the focus of research for the last decades. Today, mainly heterologous production systems are targeted due to the advantage of non-pathogenic hosts as well as uncoupling from complex quorum sensing regulatory networks compared to their natural producer Pseudomonas aeruginosa. In the recent past, the presence and function of a ROSE-like RNA-thermometer located in the 5'UTR of the rhamnosyltransferase genes rhlAB has been reported in wild type P. aeruginosa. In this study, the temperature-induced regulation of this native RNA-thermometer for heterologous rhamnolipid production was evaluated and its potential application for process control is discussed. For this purpose, the non-pathogenic production host P. putida KT2440 containing the rhlAB genes with the native P. aeruginosa 5'-UTR region was used. The system was evaluated and characterized regarding the effect of temperature on growth and product formation, as represented by efficiency parameters and yields. Experimental data suggests a major effect of temperature on specific rhamnolipid production rates. With maximum values of 0.23 g/(g h) at 37 °C, this constitutes a more than 60% increase compared to the production rate of 0.14 g/(g h) at the growth optimum of 30 °C. Interestingly however, control experiments unveiled that besides the regulatory effect of the RNA-thermometer, multiple metabolic effects may contribute equally to the observed increase in production rate. As such, this work constitutes an important step towards the utilization of temperature-based process designs and enables the possibility for novel approaches for process control.

RESUMEN

PsaA, the subunit of the fimbria originally referred to as the "pH 6 antigen," is required for full virulence of Yersinia pestis during bubonic plague. The expression of psaA is dependent upon specific environmental signals, and while the signals (high temperature and acidic pH) are defined, the mechanisms underlying this regulation remain unclear. In the closely related species Yersinia pseudotuberculosis, psaA transcription requires two regulatory genes, psaE and psaF, and it is speculated that posttranscriptional regulation of PsaE and/or PsaF contributes to the regulation of psaA transcription. Few studies have examined the regulation of psaA expression in Y. pestis, and prior to this work, the roles of psaE and psaF in Y. pestis had not been defined. The data presented here show that both psaE and psaF are required for psaA transcription in Y. pestis and that the impact of temperature and pH is mediated through discrete posttranscriptional effects on PsaE and PsaF. By generating antibodies that recognize endogenous PsaE and PsaF, we determined that the levels of both proteins are impacted by temperature and pH. High temperature is required for psaE and psaF translation via discrete mechanisms mediated by the mRNA 5' untranslated region (UTR) upstream of each gene. Additionally, levels of PsaE and PsaF are impacted by pH. We show that PsaF enhances the stability of PsaE, and thus, both PsaE and PsaF are required for psaA transcription. Our data indicate that the environmental signals (temperature and pH) impact the expression of psaA by affecting the translation of psaE and psaF and the stability of PsaE and PsaF.IMPORTANCEY. pestis is a Gram-negative bacterial pathogen that causes bubonic plague. As a vector-borne pathogen, Y. pestis fluctuates between an arthropod vector (flea) and mammalian host. As such, Y. pestis must recognize environmental signals encountered within each host environment and respond by appropriately regulating gene expression. PsaA is a key Y. pestis mammalian virulence determinant that forms fimbriae. Our work provides evidence that Y. pestis utilizes multiple posttranscriptional mechanisms to regulate the levels of two PsaA regulatory proteins in response to both temperature and pH. This study offers insight into mechanisms that bacteria utilize to sense environmental cues and regulate the expression of determinants required for mammalian disease.

Asunto(s)

RESUMEN

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5' and 3'-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.

Asunto(s)

RESUMEN

The genus Yersinia includes three human pathogenic species, Yersinia pestis, the causative agent of the bubonic and pneumonic plague, and enteric pathogens Y. enterocolitica and Y. pseudotuberculosis that cause a number of gut-associated diseases. Over the past years a large repertoire of RNA-based regulatory systems has been discovered in these pathogens using different RNA-seq based approaches. Among them are several conserved or species-specific RNA-binding proteins, regulatory and sensory RNAs as well as various RNA-degrading enzymes. Many of them were shown to control the expression of important virulence-relevant factors and have a very strong impact on Yersinia virulence. The precise targets, the molecular mechanism and their role for Yersinia pathogenicity is only known for a small subset of identified genus- or species-specific RNA-based control elements. However, the ongoing development of new RNA-seq based methods and data analysis methods to investigate the synthesis, composition, translation, decay, and modification of RNAs in the bacterial cell will help us to generate a more comprehensive view of Yersinia RNA biology in the near future.

Asunto(s)

RESUMEN

Bacterial pathogens are always challenged by fluctuations of chemical and physical parameters that pose serious threats to cellular integrity and metabolic status. Sudden deprivation of nutrients or key metabolites, changes in surrounding pH, and temperature shifts are the most important examples of such parameters. To elicit a proper response to such fluctuations, bacterial cells coordinate the expression of parameter-relevant genes. Although protein-mediated control of gene expression is well appreciated since many decades, RNA-based regulation has been discovered in early 2000s as a parallel level of regulation. Small regulatory RNAs have emerged as one of the most widespread and important gene regulatory systems in bacteria with rare representatives found in Archaea and Eukarya. Riboswitches and thermosensors are cis-encoded RNA regulatory elements that employ different mechanisms to regulate the expression of related genes controlling key metabolic pathways and genes of temperature relevant proteins including virulence factors. The extent of RNA contributions to gene regulation is not completely known even in well-studied models such E. coli and B. subtilis. In depth understanding of riboswitches is promising for opportunity to discover a narrow spectrum antibacterial drugs that target riboswitches of essential metabolic pathways.

RESUMEN

RNA structures regulate various steps in gene expression. Transcription in bacteria is typically terminated by stable hairpin structures. Translation initiation can be modulated by metabolite- or temperature-sensitive RNA structures, called riboswitches or RNA thermometers (RNATs), respectively. RNATs control translation initiation by occlusion of the ribosome binding site at low temperatures. Increasing temperatures destabilize the RNA structure and facilitate ribosome access. In this study, we exploited temperature-responsive RNAT structures to design regulatory elements that control transcription termination instead of translation initiation in Escherichia coli. In order to mimic the structure of factor-independent intrinsic terminators, naturally occurring RNAT hairpins were genetically engineered to be followed by a U-stretch. Functional temperature-responsive terminators (thermoterms) prevented mRNA synthesis at low temperatures but resumed transcription after a temperature upshift. The successful design of temperature-controlled terminators highlights the potential of RNA structures as versatile gene expression control elements.

Asunto(s)

RESUMEN

RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as Yersinia, Neisseria, and Salmonella They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5'-untranslated region (UTR) of the htrA mRNA from Salmonella enterica contains a very short RNA thermometer. We have systematically characterized the structure and dynamics of this thermometer at single-nucleotide resolution using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) assays. Our results confirm that the htrA thermometer adopts the predicted hairpin conformation at low temperatures, with conformational change occurring over a physiological temperature regime. Detailed SHAPE melting curves for individual nucleotides suggest that the thermometer unfolds in a cooperative fashion, with nucleotides from both upper and lower portions of the stem gaining flexibility at a common transition temperature. Intriguingly, analysis of an extended htrA 5' UTR sequence revealed not only the presence of the RNA thermometer, but also an additional, stable upstream structure. We generated and analyzed point mutants of the htrA thermometer, revealing elements that modulate its stability, allowing the hairpin to melt under the slightly elevated temperatures experienced during the infection of a warm-blooded host. This work sheds light on structure-function relationships in htrA and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.

Asunto(s)

RESUMEN

Like most bacteria, Shigella must maintain a precise balance between the necessity and toxicity of iron; a balance that is achieved, at least in part, by regulating the production of bacterial iron acquisition systems in response to specific environmental signals. Using the Shigella heme utilization (Shu) system, S. dysenteriae is able to acquire iron from heme, a potentially rich source of nutritional iron within the otherwise iron-limited environment of the human host. Investigations presented within reveal two distinct molecular mechanisms underlying previously uncharacterized transcriptional and translational regulation of shuT, a gene encoding the periplasmic-binding component of the Shu system. While shuT transcription is regulated in response to iron availability via a process dependent upon the global regulator Fur and a Fur-binding site located immediately downstream of the promoter, shuT translation is regulated in response to environmental temperature via the activity of an RNA thermometer located within the 5' untranslated region of the gene. Such complex regulation likely increases the fitness of S. dysenteriae by ensuring maximal ShuT production when the pathogen is within the iron-limited and relatively warm environment of the infected host, the only environment in which heme will be encountered as a potential source of essential iron.

Asunto(s)

RESUMEN

The enteric pathogen Escherichia coli O157:H7 Sakai (EHEC) is able to grow at lower temperatures compared to commensal E. coli Growth at environmental conditions displays complex challenges different to those in a host. EHEC was grown at 37°C and at 14°C with 4% NaCl, a combination of cold and osmotic stress as present in the food chain. Comparison of RNAseq and RIBOseq data provided a snap shot of ongoing transcription and translation, differentiating transcriptional and post-transcriptional gene regulation, respectively. Indeed, cold and osmotic stress related genes are simultaneously regulated at both levels, but translational regulation clearly dominates. Special emphasis was given to genes regulated by RNA secondary structures in their 5'UTRs, such as RNA thermometers and riboswitches, or genes controlled by small RNAs encoded in trans The results reveal large differences in gene expression between short-time shock compared to adaptation in combined cold and osmotic stress. Whereas the majority of cold shock proteins, such as CspA, are translationally downregulated after adaptation, many osmotic stress genes are still significantly upregulated mainly translationally, but several also transcriptionally.

Asunto(s)

RESUMEN

Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

Asunto(s)

RESUMEN

RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37 °C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25 °C, 37 °C, and 42 °C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5'-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAs.

Asunto(s)