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Berlin Brandenburg


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  • Article  (4)
  • Lucchini, Sacha  (4)
  • OneFile (GALE)  (4)
  • 1
    In: Molecular Microbiology, October 2009, Vol.74(1), pp.139-158
    Description: The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome‐wide screens in and . Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial ‘core’ small RNA, yet its function remained unknown. Here, we report that ArcZ acts as a post‐transcriptional regulator in , repressing the mRNAs of the widely distributed (serine uptake) and (oxidative stress) genes, and of STM3216, a horizontally acquired methyl‐accepting chemotaxis protein (MCP). Both and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an deletion strain further argued for the existence of a distinct set of loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady‐state levels of 〉 16% (〉 750) of all mRNAs, and rendered the bacteria non‐motile. Deep sequencing detected a dramatically changed profile of Hfq‐bound sRNAs and mRNAs, suggesting that the unprecedented pleiotropic effects by a single sRNA might in part be caused by altered post‐transcriptional regulation.
    Keywords: Biology;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 2
    In: Molecular Microbiology, May 2008, Vol.68(4), pp.890-906
    Description: Post‐transcriptional repression of porin synthesis has emerged as a major function of Hfq‐dependent, small non‐coding RNAs (sRNAs). Many enterobacteria express OmpX‐like porins, a family of outer membrane proteins whose physiological roles and structural properties have been studied intensively. While regulatory sRNAs have been identified for most major and many minor porins of and , a post‐transcriptional regulator of OmpX levels has never been found. Here, we have taken a ‘reverse target search’ approach by systematic inactivation of sRNA genes, and screening 35 sRNA deletion strains for effects on OmpX synthesis. We have identified the Hfq‐dependent CyaR (formerly RyeE) sRNA as an repressor. Global transcriptomic profiling following induction of CyaR expression suggests that mRNA is the primary target of this sRNA under standard growth conditions. The results of phylogenetic and mutational analyses suggest that a conserved RNA hairpin of CyaR, featuring a C‐rich apical loop, acts to sequester the Shine–Dalgarno sequence of mRNA and to inhibit translational initiation. We have also discovered that expression is tightly controlled by the cyclic AMP receptor protein, CRP. This represents a new link between porin repression and nutrient availability that is likely to be widely conserved among enterobacteria.
    Keywords: Genetic Research -- Genetic Aspects ; Genetic Research -- Physiological Aspects ; Bacterial Genetics -- Genetic Aspects ; Bacterial Genetics -- Physiological Aspects ; Cyclic Adenosine Monophosphate -- Genetic Aspects ; Cyclic Adenosine Monophosphate -- Physiological Aspects ; Salmonella -- Genetic Aspects ; Salmonella -- Physiological Aspects ; Rna -- Genetic Aspects ; Rna -- Physiological Aspects ; Porins -- Genetic Aspects ; Porins -- Physiological Aspects;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 3
    In: Nature Structural & Molecular Biology, 2009, Vol.16(8), p.840
    Description: Bacterial small noncoding RNAs (sRNAs) generally recognize target mRNAs in the 5' region to prevent 30S ribosomes from initiating translation. It was thought that the mRNA coding sequence (CDS) was refractory to sRNA-mediated repression, because elongating 70S ribosomes have an efficient RNA helicase activity that prevents stable target pairing. We report that the Hfq-associated MicC sRNA silences Salmonella typhimurium ompD mRNA via a [less than or equal to] 12-bp RNA duplex within the CDS (codons 23-26) that is essential and sufficient for repression. MicC does not inhibit translational initiation at this downstream position but instead acts by accelerating RNase E-dependent ompD mRNA decay. We propose an alternative gene-silencing pathway within bacterial CDS wherein sRNAs repress targets by endonucleolytic mRNA destabilization rather than by the prototypical inhibition of translational initiation. The discovery of CDS targeting markedly expands the sequence space for sRNA target predictions in bacteria.
    Keywords: Bacterial Genetics -- Research ; Gene Silencing -- Health Aspects ; Gene Silencing -- Research ; Messenger Rna -- Physiological Aspects ; Messenger Rna -- Research ; Ribosomes -- Physiological Aspects ; Ribosomes -- Research;
    ISSN: 1545-9993
    E-ISSN: 15459985
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  • 4
    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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