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.RNA structure | RNA thermometer | temperature | virulence | translational control R NA structures play a pivotal role in the function of noncoding RNAs (ncRNAs) comprised of rRNAs, tRNAs, and small regulatory RNAs (sRNAs) and in the expression of proteincoding mRNAs. Structured segments affect the entire RNA life cycle from transcription, maturation, and translation to degradation and determine the specificity of interactions with other RNAs, proteins, or ligands. Although some RNAs, such as ribozymes and rRNAs, adopt rather stable secondary and tertiary structures, many regulatory RNA elements are dynamic and undergo structural rearrangements in the physiological temperature range. Well-known examples are metabolite-sensing riboswitches (1) and temperaturesensing RNA thermometers (RNATs) (2). Bacterial RNATs are usually located in the 5′-UTR or the intercistronic region (ICR) of an mRNA and differentially control translation of the downstream ORF in response to temperature (3). Typically, an RNAT folds into a structure that occludes the ribosome binding site (RBS). A temperature upshift liberates the RBS and allows the ribosome to bind and initiate translation. Structurally diverse RNATs are located upstream of many bacterial heat shock and virulence genes. Thermosensitive RNA structures playing a role in infection and host adaptation processes have been documented in Listeria monocytogenes (4), Yersinia pestis (5), Yersinia pseudotuberculosis (6), Leptospira interrogans (7), Shigella dysenteriae (8), Neisseria meningitidis (9), Pseudomonas aeruginosa (10), and Vibrio cholerae (11).In contrast to ligand-binding riboswitches, RNATs show poor, if any, conservation in sequence and structure. This diversity has hampered the in silico identification of novel RNATs, but recently developed genome-wide RNA structure-probing approaches offer new opportunities (12). Global structure probing maps the structures of the entire pool of expressed RNA molecules and provides a sna...

Temperature variation is one of the multiple parameters a microbial pathogen encounters when it invades a warm-blooded host. To survive and thrive at host body temperature, human pathogens have developed various strategies to sense and respond to their ambient temperature. An instantaneous response is mounted by RNA thermometers (RNATs), which are integral sensory structures in mRNAs that modulate translation efficiency. At low temperatures outside the host, the folded RNA blocks access of the ribosome to the translation initiation region. The temperature shift upon entering the host destabilizes the RNA structure and thus permits ribosome binding. This reversible zipper-like mechanism of RNATs is ideally suited to fine-tune virulence gene expression when the pathogen enters or exits the body of its host. This review summarizes our present knowledge on virulence-related RNATs and discusses recent developments in the field.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37˚C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNF Y ), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25˚C but liberated at 37˚C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25˚C structure impaired opening of the stem loop, ribosome access and translation initiation at 37˚C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNF Y toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5’ untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5’ UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5’ UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi’s “stealthy” pathogenesis.

Abstract The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed ‘Lead-seq’, provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5′-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.