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Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE123048 | GEO | 2020/05/01

REPOSITORIES: GEO

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Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]

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.

2020-05-01 | GSE123049 | GEO

Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

Project description:In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay.

2019-07-08 | GSE125368 | GEO

RNase III CLASH uncovers sRNA regulatory networks coupling metabolism to production of cytolytic toxins in methicillin resistant Staphylococcus aureus

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.

2022-06-16 | PXD025122 | Pride

Small RNA interactome revealed through crosslinking of the degradosome

Project description:UV-crosslining of protein-RNA complexes was employed to capture sRNA-mRNA interactions occuring on the RNA degradosome protein, RNase E, in enterohaemorhaggic E. coli. Abstract from associated mansucript: In many organisms small regulatory RNAs (sRNA) play important roles in the regulation of gene expression by base-pairing to specific target mRNAs. In enterohaemorrhagic E. coli (EHEC), sRNAs are encoded by both the “core” genome and in numerous horizontally acquired pathogenicity islands. To identify functionally important sRNA-target RNA interactions we applied crosslinking and sequencing of hybrids (CLASH) to the core degradosome component RNase E in EHEC. RNase E was shown to bind to many classes of RNA, confirming the wide distribution of degradosome targets. These included several hundred sRNA-mRNA duplexes, and the distribution of non-templated oligo(A) tails indicated that the sRNA target RNase E-mediated cleavage at these interaction sites. Functional repression of target mRNAs was confirmed for the core sRNA RyhB, and the pathogenicity-associated sRNA Esr41. In the case of Esr41, three confirmed target mRNAs participate in iron accumulation and the ∆esr41 strain showed increased growth under conditions of iron limitation. We conclude that CLASH can be used to identify functional targets for bacterial sRNAs.

2017-02-14 | GSE77463 | GEO

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

Project description:Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

2021-09-17 | GSE139988 | GEO

Hfq Globally Binds and Destabilizes sRNAs and mRNAs in Yersinia pestis

Project description:Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3′ regions of mRNAs. An Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq-binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5′P-activated RNase E degradation pathway, which is consistent with a model in which Hfq facilitates sRNA-mRNA base pairing and the coupled degradation in Y. pestis. These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis.

2019-06-19 | GSE77555 | GEO

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression

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

A novel acetyltransferase regulates the RNA binding capacity of the RNA chaperone Hfq in Escherichia coli

Project description:Bacterial small regulatory RNAs (sRNAs) regulate gene expression by base-pairing to their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, which binds sRNAs and mRNAs on different faces. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was identified as a novel silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and determined its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppresses all tested Hfq-dependent sRNA signaling. Chromosomal HqbA suppresses the signaling of the ChiX sRNA when the C-terminus of Hfq was deleted. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked interaction also interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, suggesting that HqbA is bifunctional, with separate roles for regulating via Hfq interaction and via acetylation of undefined substrates. Gel shift assays indicated that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs, but not high-affinity RNAs. Hfq-IP RNA-Seq in WT and chromosomal hqbA mutants led to the identification of two tRNA precursors, metZWV and proM, that were enriched in Hfq binding in the absence of HqbA interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

2023-11-13 | GSE235194 | GEO

Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]

Project description:Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]

| PRJNA507415 | ENA

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus

Project description:The alpha-proteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus, and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the conserved, Hfq-bound sRNA RusT is transcriptionally controlled by the conserved NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis we determine 16 candidate target transcripts, more than half of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limiting.

2023-11-16 | GSE247928 | GEO

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