Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis

Abstract

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...

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“…This expression profile suggests that Y. pseudotuberculosis is not exposed to reactive oxygen species produced by neutrophils and other innate immune cells. However, a recent study showed that the production of multiple Yersinia catalases, superoxide dismutases, and thioredoxin (katA, sodB, sodC, and trxA) is controlled on the posttranscriptional level by thermoresponsive RNA structures (RNA thermometers) (70). They act as translational repressor elements at lower temperature but not at 37°C, and they are thus not detectable by conventional RNA-seq.…”

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“…A fruitful approach might be to use new advantages in RNA sequencing to define the secondary structure of all transcripts in a bacteria (the ‘structurome’) at one temperature and compare it with another temperature. The Narberhaus laboratory has successfully used such an approach on RNA isolated from Yersinia pseudotuberculosis to identify and characterise more than a dozen new thermosensors (Righetti et al, ). Using a similar methodology but in vivo, we have recently identified a new ‘cold sensing’ thermosensor in Listeria monocytogenes controlling expression of a cold shock protein (Ignatov and Johansson, unpublished results).…”

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“…Surprisingly, so far only one RNAT has been identified and characterized in Y. pseudotuberculosis, the lcrF RNAT located in the intergenic region of the yscWlcrF operon [42] [44]. In addition to translation, the transcription of many RNAT-controlled genes (e.g.…”

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