RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression
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ABSTRACT:
PROVIDER: PRJNA699466 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13628200_1.fastq.gz | Fastqsanger.gz | |||
| SRR13628200_2.fastq.gz | Fastqsanger.gz | |||
| SRR13628201_1.fastq.gz | Fastqsanger.gz | |||
| SRR13628201_2.fastq.gz | Fastqsanger.gz | |||
| SRR13628202_1.fastq.gz | Fastqsanger.gz |
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Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for high levels of human morbidity and mortality. MRSA co-ordinates expression of an array of bacterial factors involved in antimicrobial resistance, nutrient acquisition and immune evasion in the host. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for the control of MRSA virulence. However, the function of the majority of sRNAs during infection is unknown. To address this gap in understanding, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) in MRSA to unravel sRNA-RNA interactions under conditions that mimic the host environment. Using double stranded ribonuclease III (RNase III) as a bait we not only uncovered known interactions but also hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Importantly, we discovered that two sRNAs, RNAIII and RsaE, control the expression of at least four cytolytic toxins that are important for MRSA virulence. Taken together, we present a comprehensive analysis of sRNA-target interactions in S. aureus and provide detail on how these contribute to the control of virulence in response to changes in metabolism.
2022-05-04 | GSE166151 | GEO
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism. Regulatory small RNA (sRNA) interact with mRNAs to regulate their stability, transcription, and translation via diverse mechanisms. Here, McKellar et al. apply RNase IIICLASH of multi-drug resistant Staphylococcus aureus under different culture conditions to link the network of RNA-RNA interactions to environmental conditions and find that the production of small membrane-permeabilizing toxins is strongly regulated by sRNAs.
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Project description:Methicillin resistant Staphylococcus aureus (MRSA) is an infectious pathogen that poses a significant threat to human health. MRSA is renowned for its ability to adapt to and even thrive in hostile environment within its host. By expressing a battery of virulence factors and toxins, MRSA is able to scavenge effectively essential nutrients and evade the immune system within the host. Post-transcriptional regulation by sRNAs contributes significantly to regulating the expression of virulence factors and toxins. However, the roles of the vast majority of sRNAs during host adaptation remain unknown. To challenge this gap, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) in S. aureus to unravel sRNA-RNA interactions with the double stranded ribonuclease III (RNase III) as a bait under conditions that mimic the host environment. Here we report a global analysis of RNA-RNA interactions in MRSA in vivo, which uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results indicate that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. We show that at least two sRNAs, RNAIII and RsaE, enhance the production of five clinically relevant cytolytic toxins that are important for survival within the host. Taken together, our data greatly expands the repertoire of sRNA-target interactions in S. aureus and provide detail on how these contribute to adjusting virulence in response to changes in metabolism.
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Project description:By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.
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Project description:Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]
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Project description:Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance in poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organising maps (SOMS) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate regulation of HPr and the cell-wall autolysin Atl. These findings suggest RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment.
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