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  • Barquist, Lars  (10)
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  • 1
    Description: Program collection the data analysis of the study "Dual RNA-seq unveils noncoding RNA functions in host–pathogen interactions" by Westermann et al.,  Nature, 2016...
    Keywords: Dual Rna-Seq
    Source: DataCite
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 27 June 2017, Vol.114(26), pp.6824-6829
    Description: The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of and fully attenuates in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.
    Keywords: RNA-Binding Protein ; Salmonella ; Bacterial Pathogenesis ; Cold-Shock Protein ; Stress Response ; Bacterial Proteins ; Cold Shock Proteins and Peptides ; Heat-Shock Proteins ; RNA-Binding Proteins ; Salmonella Infections ; Salmonella Typhimurium ; Virulence Factors
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 05 November 2016, Vol.371(1707)
    Description: Infection is a complicated balance, with both pathogen and host struggling to tilt the result in their favour. Bacterial infection biology has relied on forward genetics for many of its advances, defining phenotype in terms of replication in model systems. However, many known virulence factors fail to produce robust phenotypes, particularly in the systems most amenable to genetic manipulation, such as cell-culture models. This has particularly been limiting for the study of the bacterial regulatory small RNAs (sRNAs) in infection. We argue that new sequencing-based technologies can work around this problem by providing a 'molecular phenotype', defined in terms of the specific transcriptional dysregulation in the infection system induced by gene deletion. We illustrate this using the example of our recent study of the PinT sRNA using dual RNA-seq, that is, simultaneous RNA sequencing of host and pathogen during infection. We additionally discuss how other high-throughput technologies, in particular genetic interaction mapping using transposon insertion sequencing, may be used to further dissect molecular phenotypes. We propose a strategy for how high-throughput technologies can be integrated in the study of non-coding regulators as well as bacterial virulence factors, enhancing our ability to rapidly generate hypotheses with regards to their function.This article is part of the themed issue 'The new bacteriology'.
    Keywords: Pint ; Tn-Seq ; Dual RNA-Seq ; Host–Pathogen Interaction ; Infection ; Small Non-Coding RNA ; Chromosome Mapping -- Methods ; High-Throughput Nucleotide Sequencing -- Methods ; RNA, Bacterial -- Genetics ; RNA, Small Untranslated -- Genetics ; Sequence Analysis, RNA -- Methods
    ISSN: 09628436
    E-ISSN: 1471-2970
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  • 4
    Language: English
    In: Nature, 28 January 2016, Vol.529(7587), pp.496-501
    Description: Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.
    Keywords: Gene Expression Regulation -- Genetics ; Host-Pathogen Interactions -- Genetics ; RNA, Bacterial -- Genetics ; RNA, Untranslated -- Genetics ; Salmonella Typhimurium -- Genetics
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 5
    Language: English
    In: Nucleic acids research, 02 June 2017, Vol.45(10), pp.6147-6167
    Description: Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
    Keywords: Gene Expression Regulation, Bacterial ; Transcriptome ; Bacterial Proteins -- Metabolism ; Host Factor 1 Protein -- Metabolism ; Micrornas -- Genetics ; Molecular Chaperones -- Metabolism ; Neisseria Meningitidis -- Genetics ; RNA, Bacterial -- Genetics ; RNA, Messenger -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 6
    Language: English
    In: Annual review of genetics, 2015, Vol.49, pp.367-94
    Description: Over the past decade, bacterial small RNAs (sRNAs) have gone from a biological curiosity to being recognized as a major class of regulatory molecules. High-throughput methods for sampling the transcriptional output of bacterial cells demonstrate that sRNAs are universal features of bacterial transcriptomes, are plentiful, and appear to vary extensively over evolutionary time. With ever more bacteria coming under study, the question becomes how can we accelerate the discovery and functional characterization of sRNAs in diverse organisms. New technologies built on high-throughput sequencing are emerging that can rapidly provide global insight into the numbers and functions of sRNAs in bacteria of interest, providing information that can shape hypotheses and guide research. In this review, we describe recent developments in transcriptomics (RNA-seq) and functional genomics that we expect to help us develop an integrated, systems-level view of sRNA biology in bacteria.
    Keywords: Hfq ; RNA-Seq ; Tradis ; High-Throughput Technology ; Noncoding RNA ; Phenotype Mapping ; Ribosome Profiling ; Small RNA ; RNA, Bacterial -- Analysis ; RNA, Small Untranslated -- Analysis ; Sequence Analysis, RNA -- Methods
    ISSN: 00664197
    E-ISSN: 1545-2948
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  • 7
    Language: English
    In: EMBO journal: European Molecular Biology Organization, 2016, Issue 9, pp.991-1011
    Description: The molecular roles of many RNA‐binding proteins in bacterial post‐transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP‐seq) have begun to revolutionize the transcriptome‐wide mapping of eukaryotic RNA‐binding protein target sites. We have applied CLIP‐seq to chart the target landscape of two major bacterial post‐transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single‐nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3′‐located Rho‐independent terminators as a universal motif involved in Hfq–RNA interactions. Additionally, Hfq preferentially binds 5′ to sRNA‐target sites in mRNAs, and 3′ to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA–mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA‐target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP‐seq approach will bring new insights into post‐transcriptional gene regulation by RNA‐binding proteins in diverse bacterial species.
    Keywords: Clip ; Csra ; Hfq ; Non‐Coding Rna ; Peak Calling ; Post‐Transcriptional Control ; Small Rna ; Terminator ; Translation
    ISSN: 0261-4189
    Source: Fundación Dialnet
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  • 8
    In: EMBO Journal, 02 May 2016, Vol.35(9), pp.991-1011
    Description: The molecular roles of many ‐binding proteins in bacterial post‐transcriptional gene regulation are not well understood. Approaches combining crosslinking with deep sequencing (‐seq) have begun to revolutionize the transcriptome‐wide mapping of eukaryotic ‐binding protein target sites. We have applied ‐seq to chart the target landscape of two major bacterial post‐transcriptional regulators, Hfq and CsrA, in the model pathogen Typhimurium. By detecting binding sites at single‐nucleotide resolution, we identify preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3′‐located Rho‐independent terminators as a universal motif involved in Hfq– interactions. Additionally, Hfq preferentially binds 5′ to ‐target sites in s, and 3′ to seed sequences in s, reflecting a simple logic in how Hfq facilitates – interactions. Importantly, global knowledge of Hfq sites significantly improves ‐target predictions. CsrA binds sequences in apical loops and targets many virulence s. Overall, our generic ‐seq approach will bring new insights into post‐transcriptional gene regulation by ‐binding proteins in diverse bacterial species. A new pipeline for ‐seq in maps global –protein interactions and offers a tool for improved understanding of post‐transcriptional control in bacteria. Transcriptome‐wide mapping of Hfq and CsrA target sites by CLIP‐seq. Rho‐independent terminators comprise a general Hfq‐binding motif. Hfq binds 5′ to sRNA‐binding sites in mRNA targets and 3′ to seed sequences in cognate the sRNAs. CsrA preferentially recognizes AUGGA sequences present in loops of hairpin structures. CsrA binds and regulates many mRNAs encoding virulence factors. A new pipeline for CLIP‐seq in maps global RNA–protein interactions and offers a tool for improved understanding of post‐transcriptional control in bacteria.
    Keywords: Clip ; Csra ; Hfq ; Non‐Coding Rna ; Peak Calling ; Post‐Transcriptional Control ; Small Rna ; Terminator ; Translation
    ISSN: 0261-4189
    E-ISSN: 1460-2075
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  • 9
    In: PLoS Pathogens, 2017, Vol.13(2)
    Description: The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.
    Keywords: Review ; Biology And Life Sciences ; Research And Analysis Methods ; Medicine And Health Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Medicine And Health Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Research And Analysis Methods
    ISSN: 1553-7366
    E-ISSN: 1553-7374
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  • 10
    Language: English
    In: Molecular Cell, 07 June 2018, Vol.70(5), pp.971-982.e6
    Description: The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in and . Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3′ end of mRNAs. Using the mRNA as a model for 3′ end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs. Using CLIP-seq, Holmqvist et al. map transcriptome-wide interactions of the emerging global RNA-binding protein ProQ in and . Their data suggest ProQ to target sRNAs and mRNA 3′ UTRs primarily through a structural code and to stabilize some mRNAs by counteracting 3′ exoribonuclease activity.
    Keywords: Proq ; Clip-Seq ; RNA-Binding Protein ; 3′ Utr ; Post-Transcriptional Control ; Exoribonuclease ; Biology
    ISSN: 1097-2765
    E-ISSN: 1097-4164
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