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  • 1
    Language: English
    In: PLoS ONE, 2011, Vol.6(3), p.e17296
    Description: P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella -induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri , arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Microbiology ; Molecular Biology ; Cell Biology
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PLoS Genetics, 2012, Vol.8(6), p.e1002782
    Description: RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus . However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo . Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. ; Control of mRNA stability is crucial for bacteria to survive and rapidly adapt to environmental changes and stress conditions. The molecular players and the degradation pathways involved in these adaptive processes are poorly understood in . The universally conserved double-strand-specific endoribonuclease III (RNase III) in is known to repress the synthesis of several virulence factors and was recently implicated in genome-wide mRNA processing mediated by antisense transcripts. We present here the first global map of direct RNase III targets in . Deep sequencing was used to identify RNAs associated with epitope-tagged wild-type RNase III and two catalytically impaired but binding-competent mutant proteins . Experimental validation revealed an unexpected variety of structured RNA transcripts as novel RNase III substrates. In addition to rRNA operon maturation, autoregulation, degradation of structured RNAs, and antisense regulation, we propose novel mechanisms by which RNase III increases mRNA translation. Overall, this study shows that RNase III has a broad function in gene regulation of . We can now address more specifically the roles of this universally conserved enzyme in gene regulation in response to stress and during host infection.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 3
    Language: English
    In: PLoS Biology, 2008, Vol.6(3), p.e64
    Description: Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3′ end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms. ; Hierarchical action of regulators is a fundamental principle in gene expression control, and is well understood in protein-based signaling pathways. We have discovered that small noncoding RNAs (sRNAs), a new class of gene expression regulators, can also act hierarchically and form a regulatory cascade. Two highly similar sRNAs function after transcription to activate the mRNA, which codes for an essential function in amino-sugar metabolism. It is somewhat unusual for two sRNAs to act upon the same target mRNA, and despite their seeming homology, these two sRNAs (GlmY and GlmZ) employ different molecular mechanisms and function hierarchically to activate expression: GlmZ directly activates translation via disruption of an mRNA structure that inhibits translation, whereas GlmY controls the processing of GlmZ to prevent the inactivation of this direct activator. We also found that GlmY is itself controlled by an RNA processing event (3′ end polyadenylation), which typically destabilizes bacterial RNA. Our data unequivocally demonstrate that is exceptionally dependent on RNA-based mechanisms for its genetic control. Given the large number of noncoding RNAs of unknown function, we believe that similar regulatory RNA cascades may operate in other organisms. ; A regulatory RNA cascade that posttranscriptionally activates the mRNA is identified, with two highly similar small noncoding RNAs acting hierarchically in a manner thus far known only in protein-based regulatory circuits.
    Keywords: Research Article ; Biochemistry ; Genetics And Genomics ; Molecular Biology
    ISSN: 1544-9173
    E-ISSN: 1545-7885
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  • 4
    Language: English
    In: PLoS Computational Biology, 2009, Vol.5(9), p.e1000502
    Description: With few exceptions, current methods for short read mapping make use of simple seed heuristics to speed up the search. Most of the underlying matching models neglect the necessity to allow not only mismatches, but also insertions and deletions. Current evaluations indicate, however, that very different error models apply to the novel high-throughput sequencing methods. While the most frequent error-type in Illumina reads are mismatches, reads produced by 454's GS FLX predominantly contain insertions and deletions (indels). Even though 454 sequencers are able to produce longer reads, the method is frequently applied to small RNA (miRNA and siRNA) sequencing. Fast and accurate matching in particular of short reads with diverse errors is therefore a pressing practical problem. We introduce a matching model for short reads that can, besides mismatches, also cope with indels. It addresses different error models. For example, it can handle the problem of leading and trailing contaminations caused by primers and poly-A tails in transcriptomics or the length-dependent increase of error rates. In these contexts, it thus simplifies the tedious and error-prone trimming step. For efficient searches, our method utilizes index structures in the form of enhanced suffix arrays. In a comparison with current methods for short read mapping, the presented approach shows significantly increased performance not only for 454 reads, but also for Illumina reads. Our approach is implemented in the software segemehl available at http://www.bioinf.uni-leipzig.de/Software/segemehl/ . ; The successful mapping of high-throughput sequencing (HTS) reads to reference genomes largely depends on the accuracy of both the sequencing technologies and reference genomes. Current mapping algorithms focus on mapping with mismatches but largely neglect insertions and deletions—regardless of whether they are caused by sequencing errors or genomic variation. Furthermore, trailing contaminations by primers and declining read qualities can be cumbersome for programs that allow a maximum number of mismatches. We have developed and implemented a new approach for short read mapping that, in a first step, computes exact matches of the read and the reference genome. The exact matches are then modified by a limited number of mismatches, insertions and deletions. From the set of exact and inexact matches, we select those with minimum score-based E-values. This gives a set of regions in the reference genome which is aligned to the read using Myers bitvector algorithm . Our method utilizes enhanced suffix arrays to quickly find the exact and inexact matches. It maps more reads and achieves higher recall rates than previous methods. This consistently holds for reads produced by 454 as well as Illumina sequencing technologies.
    Keywords: Research Article ; Computational Biology ; Computational Biology -- Genomics ; Genetics And Genomics -- Bioinformatics
    ISSN: 1553-734X
    E-ISSN: 1553-7358
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  • 5
    Language: English
    In: PLoS Genetics, 2008, Vol.4(8), p.e1000163
    Description: Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo . The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella . Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria. ; The past decade has seen small regulatory RNA become an important new mediator of bacterial mRNA regulation. This study describes a rapid way to identify novel sRNAs that are expressed, and should prove relevant to a variety of bacteria. We purified the epitope-tagged RNA-binding protein, Hfq, and its bound RNA by immunoprecipitation from the model pathogen, serovar Typhimurium. This new strategy used Next Generation pyrosequencing to identify 727 Hfq-bound mRNAs. The numbers of sRNAs expressed in was doubled to 64; half are associated with Hfq. We defined the exact coordinates of sRNAs, and confirmed that they are expressed at significant levels. We also determined the Hfq regulon in , and reported the role of Hfq in controlling transcription of major pathogenicity islands, horizontally acquired regions, and the flagellar cascade. Hfq is reported to be a global regulator that affects the expression of almost a fifth of all genes. Our new approach will allow sRNAs and mRNAs to be characterized from different genetic backgrounds, or from bacteria grown under particular environmental conditions. It will be valuable to scientists working on genetically tractable bacteria who are interested in the function of RNA-binding proteins and the identification of sRNAs.
    Keywords: Research Article ; Biochemistry -- Bioinformatics ; Genetics And Genomics -- Functional Genomics ; Genetics And Genomics -- Gene Expression ; Microbiology ; Microbiology -- Microbial Evolution And Genomics
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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