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Berlin Brandenburg


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  • RNA-Binding Proteins  (7)
  • Hfq
Type of Medium
  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 11 October 2016, Vol.113(41), pp.11591-11596
    Description: The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.
    Keywords: Hfq ; Proq ; RNA–Protein Interaction ; Noncoding RNA ; Small RNA ; Bacterial Proteins -- Metabolism ; High-Throughput Nucleotide Sequencing -- Methods ; RNA, Bacterial -- Metabolism ; RNA-Binding Proteins -- Metabolism ; Salmonella Enterica -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Frontiers in cellular and infection microbiology, 2014, Vol.4, pp.91
    Description: Enteric pathogens often cycle between virulent and saprophytic lifestyles. To endure these frequent changes in nutrient availability and composition bacteria possess an arsenal of regulatory and metabolic genes allowing rapid adaptation and high flexibility. While numerous proteins have been characterized with regard to metabolic control in pathogenic bacteria, small non-coding RNAs have emerged as additional regulators of metabolism. Recent advances in sequencing technology have vastly increased the number of candidate regulatory RNAs and several of them have been found to act at the interface of bacterial metabolism and virulence factor expression. Importantly, studying these riboregulators has not only provided insight into their metabolic control functions but also revealed new mechanisms of post-transcriptional gene control. This review will focus on the recent advances in this area of host-microbe interaction and discuss how regulatory small RNAs may help coordinate metabolism and virulence of enteric pathogens.
    Keywords: Csra ; Hfq ; Carbon Metabolism ; Srna ; Virulence ; Energy Metabolism ; Carbon -- Metabolism ; Intestines -- Microbiology ; RNA, Small Untranslated -- Genetics
    E-ISSN: 2235-2988
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  • 3
    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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  • 4
    Language: English
    In: RNA Biology, 01 April 2012, Vol.9(4), pp.520-531
    Description: Helicobacter pylori, one of the most prevalent human pathogens, used to be thought to lack small regulatory RNAs (sRNAs) which are otherwise considered abundant in all bacteria. However, our recent analysis of the primary transcriptome of H. pylori discovered an unexpectedly large number of...
    Keywords: RNA-Seq ; Small RNA ; Hfq ; Helicobacter Pylori ; RNA Binding Proteins ; Affinity Chromatography ; Post-Transcriptional Control ; Co-Immunoprecipitation ; Anatomy & Physiology
    ISSN: 1547-6286
    E-ISSN: 1555-8584
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  • 5
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2012, Vol.905, pp.177-200
    Description: Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.
    Keywords: Aptamers, Nucleotide -- Metabolism ; RNA, Small Untranslated -- Metabolism ; RNA-Binding Proteins -- Metabolism
    ISBN: 9781617799488
    ISSN: 10643745
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 6
    Language: English
    In: Biological chemistry, December 2005, Vol.386(12), pp.1219-38
    Description: Small non-coding RNAs (sRNAs) have attracted considerable attention as an emerging class of gene expression regulators. In bacteria, a few regulatory RNA molecules have long been known, but the extent of their role in the cell was not fully appreciated until the recent discovery of hundreds of potential sRNA genes in the bacterium Escherichia coli. Orthologs of these E. coli sRNA genes, as well as unrelated sRNAs, were also found in other bacteria. Here we review the disparate experimental approaches used over the years to identify sRNA molecules and their genes in prokaryotes. These include genome-wide searches based on the biocomputational prediction of non-coding RNA genes, global detection of non-coding transcripts using microarrays, and shotgun cloning of small RNAs (RNomics). Other sRNAs were found by either co-purification with RNA-binding proteins, such as Hfq or CsrA/RsmA, or classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels. In addition, bacterial genetics offers powerful tools that aid in the search for sRNAs that may play a critical role in the regulatory circuit of interest, for example, the response to stress or the adaptation to a change in nutrient availability. Many of the techniques discussed here have also been successfully applied to the discovery of eukaryotic and archaeal sRNAs.
    Keywords: Gene Expression Regulation, Bacterial ; RNA, Bacterial ; RNA, Untranslated ; RNA-Binding Proteins
    ISSN: 1431-6730
    E-ISSN: 14374315
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  • 7
    In: mBio, 2019, Vol.10(1)
    Description: The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. ABSTRACT FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella , Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.
    Keywords: Research Article ; Molecular Biology And Physiology ; Editor'S Pick ; Hfq ; Noncoding Rna ; Proq ; Rna-Seq ; Bacterial Pathogen ; Posttranscriptional Control
    ISSN: 21612129
    E-ISSN: 2150-7511
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