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  • 1.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 2.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 3. Avesson, Lotta
    et al.
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart Eduard Heinrich
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Söderbom, Fredrik
    Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs2012Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, nr 10, s. 1771-1782Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a common origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. One example is miRNAs. Their regulation of protein coding genes has been shown to play a vital role in plants and animals but little is known about their role in other organisms. The single cell social amoeba Dictyostelium discoideum could hold the answers to some questions regarding the evolution and function of small RNA pathways. Here we report deep sequencing of small RNAs from three developmental stages of D. discoideum. Analyses of these libraries as well as experimental data reveal the expression of a number of miRNAs, several which have distinct expression patterns during development. We also find miRNAs processed from a hairpin originating from a repetitive element that we believe could represent a pathway for the generation of new miRNAs.

  • 4.
    Baranowska, Izabella
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Jäderlund, Karin Hultin
    Nennesmo, Inger
    Holmqvist, Erik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Heidrich, Nadja
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Larsson, Nils-Göran
    Andersson, Göran
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hedhammar, Åke
    Wibom, Rolf
    Andersson, Leif
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene2009Ingår i: PLoS Genetics, ISSN 1553-7390, Vol. 5, nr 5, s. e1000499-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.

  • 5.
    Bass, Brenda L.
    et al.
    Univ Utah, Dept Biochem, Salt Lake City, UT 84112 USA.
    O'Connell, Mary A.
    CEITEC Masaryk Univ, Brno 62500, Czech Republic.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Biographical item: "A celebration of the life of Marie Öhman (1964-2019)" in RNA, Volume: 25 Issue: 10, Pages: IX-XI2019Övrigt (Övrig (populärvetenskap, debatt, mm))
  • 6.
    Berghoff, Bork A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Aulbach, Lena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Two regulatory RNA elements affect TisB-dependent depolarization and persister formation2017Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, nr 6, s. 1020-1033Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.

  • 7.
    Berghoff, Bork A.
    et al.
    Justus Liebig Univ, Inst Mikrobiol & Mol Biol, Giessen, Germany..
    Karlsson, Torgny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk genetik och genomik.
    Kallman, Thomas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Grabherr, Manfred G.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    RNA-sequence data normalization through in silico prediction of reference genes: the bacterial response to DNA damage as case study2017Ingår i: BioData Mining, ISSN 1756-0381, E-ISSN 1756-0381, Vol. 10, artikel-id 30Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Measuring how gene expression changes in the course of an experiment assesses how an organism responds on a molecular level. Sequencing of RNA molecules, and their subsequent quantification, aims to assess global gene expression changes on the RNA level (transcriptome). While advances in high-throughput RNA-sequencing (RNA-seq) technologies allow for inexpensive data generation, accurate post-processing and normalization across samples is required to eliminate any systematic noise introduced by the biochemical and/or technical processes. Existing methods thus either normalize on selected known reference genes that are invariant in expression across the experiment, assume that the majority of genes are invariant, or that the effects of up-and down-regulated genes cancel each other out during the normalization.

    Results: Here, we present a novel method, moose(2), which predicts invariant genes in silico through a dynamic programming (DP) scheme and applies a quadratic normalization based on this subset. The method allows for specifying a set of known or experimentally validated invariant genes, which guides the DP. We experimentally verified the predictions of this method in the bacterium Escherichia coli, and show how moose(2) is able to (i) estimate the expression value distances between RNA-seq samples, (ii) reduce the variation of expression values across all samples, and (iii) to subsequently reveal new functional groups of genes during the late stages of DNA damage. We further applied the method to three eukaryotic data sets, on which its performance compares favourably to other methods. The software is implemented in C++ and is publicly available from http://grabherr.github.io/moose2/.

    Conclusions: The proposed RNA-seq normalization method, moose(2), is a valuable alternative to existing methods, with two major advantages: (i) in silico prediction of invariant genes provides a list of potential reference genes for downstream analyses, and (ii) non-linear artefacts in RNA-seq data are handled adequately to minimize variations between replicates.

  • 8.
    Berghoff, Bork A.
    et al.
    Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence2017Ingår i: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 63, nr 6, s. 1011-1016Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Bacterial dormancy is a valuable survival strategy upon challenging environmental conditions. Dormant cells tolerate the consequences of high stress levels and may re-populate the environment upon return to favorable conditions. Antibiotic-tolerant bacteria-termed persisters-regularly cause relapsing infections, increase the likelihood of antibiotic resistance, and, therefore, earn increasing attention. Their generation often depends on toxins from chromosomal toxin-antitoxin systems. Here, we review recent insights concerning RNA-based control of toxin synthesis, and discuss possible implications for persister generation.

  • 9.
    Darfeuille, Fabien
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Unoson, Cecilia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Vogel, Jörg
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    An antisense RNA inhibits translation by competing with standby ribosomes2007Ingår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 26, nr 3, s. 381-392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or “standby” site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located 100 nucleotides upstream.

  • 10.
    Di Martino, Maria Letizia
    et al.
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    Romilly, Cedric
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Colonna, Bianca
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    Prosseda, Gianni
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator2016Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, nr 6, artikel-id e01860-16Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF(30) (30 kDa), and the shorter VirF(21) (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF(30) and VirF(21) and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF(21) is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF(21). The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF(30) is responsible for activation of the virulence system, VirF(21) negatively autoregulates virF expression itself. Since VirF(21) modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF(30) and VirF(21), that are functionally distinct. The major form, VirF(30), activates the genes necessary for virulence, whereas the minor VirF(21), which shares the C-terminal two-thirds of VirF(30), negatively autoregulates virF expression itself. VirF(21) is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.

  • 11. Djupedal, Ingela
    et al.
    Kos-Braun, C
    Mosher, A
    Söderholm, Niklas
    Simmer, Femke
    Hardcastle, J
    Fender, Aurelie
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Heidrich, Nadja
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kagansky, Alexander
    Bayne, Elizabeth
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Baulcombe, C
    Allshire, C
    Ekwall, Karl
    Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA2009Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, nr 24, s. 3832-3844Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009

  • 12. Engdahl, Hilde
    et al.
    Lindell, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart
    Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation2001Ingår i: Antisense Nucleic Acid Drug Dev, Vol. 11, nr 1, s. 29-40Artikel i tidskrift (Refereegranskat)
  • 13.
    Fender, Aurelie
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Elf, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Hampel, Kornelia
    Zimmermann, Bastian
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    RNAs actively cycle on the Sm-like protein Hfq2010Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 24, nr 23, s. 2621-2626Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K-d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are approximate to 1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the "strong binding-high turn-over" paradox and permits efficient use of the Hfq pool.

  • 14. Gerdes, Kenn
    et al.
    Wagner, Gerhart H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    RNA antitoxins2007Ingår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 10, nr 2, s. 117-124Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Recent genomic analyses revealed a surprisingly large number of toxin–antitoxin loci in free-living prokaryotes. The antitoxins are proteins or antisense RNAs that counteract the toxins. Two antisense RNA-regulated toxin–antitoxin gene families, hok/sok and ldr, are unrelated sequence-wise but have strikingly similar properties at the level of gene and RNA organization. Recently, two SOS-induced toxins were found to be regulated by RNA antitoxins. One such toxin, SymE, exhibits similarity with MazE antitoxin and, surprisingly, inhibits translation. Thus, it is possible that an ancestral antitoxin gene evolved into the present toxin gene (symE) whose translation is repressed by an RNA antitoxin (SymR).

  • 15. Hammann, Philippe
    et al.
    Parmentier, Delphine
    Cerciat, Marie
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    Helfer, Anne-Catherine
    Boisset, Sandrine
    Guillier, Maude
    Vandenesch, Francois
    Wagner, Gerhart H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Romby, Pascale
    Fechter, Pierre
    A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus2014Ingår i: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 106C, s. 175-179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia colt, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.

  • 16. He, Lin
    et al.
    Söderbom, Fredrik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart
    Binnie, Uta
    Binns, Nigel
    Masters, Millicent
    PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids1993Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 9, nr 6, s. 1131-1142Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

  • 17.
    Hinas, Andrea
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Nellen, Wolfgang
    Ambros, Victor R.
    Söderbom, Fredrik
    The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway2007Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, nr 20, s. 6714-6726Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 1826 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.

  • 18.
    Hoekzema, Mirthe
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Romilly, Cedric
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Holmqvist, Erik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation2019Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, artikel-id e101199Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.

  • 19.
    Holmqvist, Erik
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Reimegard, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing2013Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, nr 12, s. e122-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.

  • 20.
    Holmqvist, Erik
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Unoson, Cecilia
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp2012Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 84, nr 3, s. 414-427Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.

  • 21.
    Holmqvist, Erik
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Unoson, Cecilia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    The sRNA MicF targets its own regulator Lrp and promotes a positive feedback loopManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    As much as 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, whose expression changes depending on the nutritional status of the environment. The output of Lrp regulation can be modulated by cellular leucine, which either enhances or reverses the effect on Lrp-targeted promoters. In a bioinformatic search for sRNA targets, lrp was identified as a putative target for the MicF sRNA, whose expression is negatively regulated by Lrp. A deletion of micF resulted in higher Lrp levels, while overexpression of MicF strongly inhibited Lrp expression. Mutations in the predicted interaction sequence of MicF and lrp relieved MicF-dependent repression of Lrp synthesis, both in vivo and in vitro. The predicted base-pairing interaction was supported by structural probing. Additionally, we show that MicF specifically interferes with initiating ribosomes on the lrp mRNA in vitro. In vivo, MicF-dependent inhibition of Lrp synthesis resulted in increased expression of the livJ gene, a member of the Lrp regulon. Finally, MicF was shown to increase its own expression through Lrp, creating a positive feedback loop. These findings contribute to the understanding of Lrp regulation in particular and the involvement of sRNAs in regulatory networks in general.

  • 22.
    Holmqvist, Erik
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, E. Gerhart H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Impact of bacterial sRNAs in stress responses2017Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 45, s. 1203-1212Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Bacterial life is harsh and involves numerous environmental and internal challenges that are perceived as stresses. Consequently, adequate responses to survive, cope with, and counteract stress conditions have evolved. In the last few decades, a class of small, non-coding RNAs (sRNAs) has been shown to be involved as key players in stress responses. This review will discuss - primarily from an enterobacterial perspective - selected stress response pathways that involve antisense-type sRNAs. These include themes of how bacteria deal with severe envelope stress, threats of DNA damage, problems with poisoning due to toxic sugar intermediates, issues of iron homeostasis, and nutrient limitation/starvation. The examples discussed highlight how stress relief can be achieved, and how sRNAs act mechanistically in regulatory circuits. For some cases, we will propose scenarios that may suggest why contributions from post-transcriptional control by sRNAs, rather than transcriptional control alone, appear to be a beneficial and universally selected feature.

  • 23.
    Ivanova, Natalia
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lindell, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Holmberg Schiavone, Lovisa
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Structure probing of tmRNA in distinct stages of trans-translation2007Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, nr 5, s. 713-722Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was                  d      dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

  • 24.
    Lindell, Magnus
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Mikrobiologi.
    Brännvall, Mathias
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Mikrobiologi.
    Kirsebom, Leif A
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Mikrobiologi.
    Lead(II) cleavage analysis of RNase P RNA in vivo.2005Ingår i: RNA, ISSN 1355-8382, Vol. 11, nr 9, s. 1348-54Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.

  • 25.
    Lindell, Magnus
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Brännvall, Mathias
    Wagner, Gerhart
    Kirsebom, Leif
    Lead(II) cleavage analysis of RNase P RNA in vivoArtikel i tidskrift (Refereegranskat)
  • 26.
    Lindell, Magnus
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Romby, Pascale
    Wagner, Gerhart
    Lead(II) as a probe for investigating RNA structure in vivo2002Ingår i: RNA, Vol. 8, nr 4, s. 534-541Artikel i tidskrift (Refereegranskat)
  • 27.
    Melin, Petter
    et al.
    WAGNER GROUP.
    Schnürer, Johan
    Wagner, Gerhart
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Disruption of the gene encoding the V-ATPase subunit A results in inhibition of normal growth and abolished sporulation in Aspergillus nidulans2003Ingår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 150, nr 3, s. 743-748Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn2+ concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.

  • 28. Park, Hyun-Sook
    et al.
    Ostberg, Yngve
    Johansson, Jorgen
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Uhlin, Bernt Eric
    Novel role for a bacterial nucleoid protein in translation of mRNAs with suboptimal ribosome-binding sites2010Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 24, nr 13, s. 1345-1350Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In Escherichia coli, the major nucleoid protein H-NS limits transcription by acting as a repressor or transcriptional silencer, presumably by its ability to close the looped chromosome domains in the nucleoid through DNA-protein-DNA bridging. Here, we demonstrate the direct involvement of H-NS as a positive factor stimulating translation of the malT mRNA. In vitro studies showed that H-NS facilitates a repositioning of the 30S preinitiation complex on the malT mRNA. H-NS stimulation of translation depended on the AU-rich -35 to -40 region of the mRNA. Several additional examples were found demonstrating a novel function for H-NS in translation of genes with suboptimal ribosome-binding sequences.

  • 29. Quax, Tessa E. F.
    et al.
    Wolf, Yuri I.
    Koehorst, Jasper J.
    Wurtzel, Omri
    van der Oost, Richard
    Ran, Wenqi
    Blombach, Fabian
    Makarova, Kira S.
    Brouns, Stan J. J.
    Forster, Anthony C.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Wagner, Gerhart
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Sorek, Rotem
    Koonin, Eugene V.
    van der Oost, John
    Differential Translation Tunes Uneven Production of Operon-Encoded Proteins2013Ingår i: Cell Reports, ISSN 2211-1247, Vol. 4, nr 5, s. 938-944Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

  • 30. Reimegård, Johan
    et al.
    Ardell, David
    Wagner, Gerhart Eduard Heinrich
    AntisenseRNA: fast, specific target prediction for bacterial sRNAs through models of interaction and evolutionary conservationManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Small regulatory RNAs (sRNAs) are ubiquitously present in many different bacteria and are often conserved in closely related species. The majority of the sRNAs acts as antisense RNAs via basepairing to target mRNAs and mediates up- or down-regulation. This involves effects on translation and/or mRNA stability. Since antisense-target RNA interaction sites are often short and non-contiguous, prediction of targets is a non-trivial task. Thus, most sRNA have not yet been assigned to specific target genes, and this motivates a need for computational target prediction programs. Available programs use criteria such as, for instance, a "seed" antisense-target interaction and the structural accessibility of the interaction site to indentify sRNA targets. So far, the realization that RNA/RNA interaction is a hierarchical multi-step process has not been incorporated into algorithms, and neither has conservation of interactions between related species. We have designed a target prediction program, AntisenseRNA, which considers the interaction as a multistep process, in which the intramolecular structure of the two RNAs and the phylogenetic conservation of basepairs are taken into account. AntisenseRNA successfully identifies most of the experimentally validated sRNA targets in Escherichia coli and predicts their interaction sites with high specificity and sensitivity. AntisenseRNA is about ten times faster than its best competitor programs, suggesting it to be a good choice for identification of new targets for sRNAs.

  • 31. Romby, Pascale
    et al.
    Vandenesch, Francois
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. mikrobiologi.
    The role of RNAs in the regulation of virulence-gene expression.2006Ingår i: Curr Opin Microbiol, ISSN 1369-5274, Vol. 9, nr 2, s. 229-36Artikel i tidskrift (Refereegranskat)
  • 32. Romby, Pascale
    et al.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Exploring the complex world of RNA regulation.2008Ingår i: Biology of the Cell, ISSN 0248-4900, E-ISSN 1768-322X, Vol. 100, nr 1, s. e1-3Artikel i tidskrift (Refereegranskat)
  • 33.
    Romilly, Cedric
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Deindl, Sebastian
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element2019Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, nr 32, s. 15901-15906Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30 Delta S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (similar to 100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

  • 34.
    Sterk, Maaike
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Romilly, Cedric
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 8, s. 4188-4199Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails.

  • 35.
    Timmusk, Salme
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Grantcharova, Nina
    Flärdh, Klas
    Wagner, Gerhart E. H.
    Paenibacillus polymyxa colonization of Arabidopsis thaliana rootsIngår i: AEMArtikel i tidskrift (Refereegranskat)
  • 36.
    Timmusk, Salme
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. mikrobiologi.
    Grantcharova, Nina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. mikrobiologi.
    Paenibacillus polymyxa Invades Plant Roots and Forms Biofilms.2005Ingår i: Appl Environ Microbiol, ISSN 0099-2240, Vol. 71, nr 11, s. 7292-300Artikel i tidskrift (Övrigt vetenskapligt)
  • 37.
    Timmusk, Salme
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    van West, Pieter
    Gow, Neil A. R.
    Wagner, Gerhart E. H.
    Antagonistic effects of Paenibacillus polymyxa towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatumManuskript (Övrigt vetenskapligt)
  • 38.
    Timmusk, Salme
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart E. H.
    The plant-growth-promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: a possible connection between biotic and abiotic stress responses1999Ingår i: MPMI, Vol. 12, nr 11, s. 951-959Artikel i tidskrift (Refereegranskat)
  • 39.
    Udekwu, Klas I
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Darfeuille, Fabien
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Vogel, Jörg
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Holmqvist, Erik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart. E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hfq-dependent regulation of OmpA synthesis is mediated by an an-tisense RNA.2005Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 19, nr 19, s. 2355-2366Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which—in line with previous work—secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.

  • 40.
    Udekwu, Klas I.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Sigma E controls biogenesis of the antisense RNA MicA2007Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, nr 4, s. 1279-1288Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σS. We identified stress conditions that induce high MicA levels even during exponential growth - a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σE is activated. The strict dependence of micA transcription on σE is supported by three observations. Induced overexpression of σE increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σE signature. Thus, MicA is part of the σE regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.

  • 41.
    Udekwu, Klas I.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart H.
    The Escherichia coli luxS mRNA is processed in an RNase III- dependent fashion inManuskript (Övrigt vetenskapligt)
  • 42.
    Unoson, Cecilia
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli2008Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, nr 1, s. 258-70Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We previously reported on an SOS-induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR-1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.

  • 43.
    Unoson, Cecilia
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Dealing with stable structures at ribosome binding sites: bacterial translation and ribosome standby.2007Ingår i: RNA Biology, ISSN 1547-6286, Vol. 4, nr 3, s. 113-117Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.

  • 44. Valverde, Claudio
    et al.
    Lindell, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Haas, Dieter
    A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens2004Ingår i: J. Biol. Chem., Vol. 279, s. 25066-25074Artikel i tidskrift (Refereegranskat)
  • 45. Valverde, Claudio
    et al.
    Lindell, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart
    Haas, Dieter
    A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens2004Ingår i: J Biol Chem, Vol. 279, nr 24, s. 25066-25074Artikel i tidskrift (Refereegranskat)
  • 46.
    Vogel, Jörg
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. mikrobiologi.
    Argaman, Liron
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. mikrobiologi.
    Altuvia, Shoshy
    The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide2004Ingår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 14, nr 24, s. 2271-2276Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    More than 60 small RNAs (sRNA) have been identified in E. coli 1, 2, 3, 4, 5, 6 and 7. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions 8, 9, 10, 11, 12, 13 and 14. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

  • 47.
    Vogel, Jörg
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. mikrobiologi.
    Bartels, Verena
    Tang, Thean Hock
    Churakov, Gennady
    Slagter-Jäger, Jacoba G
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hüttenhofer, Alexander
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. mikrobiologi.
    RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria2003Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, nr 22, s. 6435-6443Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent bioinformatics‐aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot‐gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co‐expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III‐dependent cleavage. To the best of our knowledge, this represents the first case of two non‐coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half‐lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.

  • 48. Vogel, Jörg
    et al.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Target identification of small noncoding RNAs in bacteria2007Ingår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 10, nr 3, s. 262-270Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Small noncoding RNAs have been discovered at a staggering rate in Escherichia coli and many other bacteria. Most of the sRNAs of known function regulate gene expression by binding to specific mRNAs or proteins. Given the scores of sRNAs of unknown function, the identification of their cellular targets has become urgent. Here, we review the diverse strategies that have been used to identify and validate bacterial sRNA targets. These include the pulse-expression of sRNAs followed by global transcriptome analysis (microarrays), new biocomputational prediction algorithms, and novel gfp reporter gene fusions to validate candidate target gene regulation.

  • 49.
    Wagner, Gerhart
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Cycling of RNAs on Hfq2013Ingår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, nr 4, s. 619-626Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The RNA chaperone Hfq is a key player in small RNA (sRNA)-mediated regulation of target mRNAs in many bacteria. The absence of this protein causes pleiotropic phenotypes such as impaired stress regulation and, occasionally, loss of virulence. Hfq promotes rapid sRNA-target mRNA base pairing to allow for fast, adaptive responses. For this to happen, sRNAs and/or mRNAs must be bound by Hfq. However, when the intra- or extracellular environment changes, so does the intracellular RNA pool, and this, in turn, requires a correspondingly rapid change in the pool of Hfq-bound RNAs. Biochemical studies have suggested tight binding of Hfq to many RNAs, indicating very slow dissociation rates. In contrast, the changing pool of binding-competent RNAs must compete for access to this helper protein in a minute time frame (known response time for regulation). How rapid exchange of RNAs on Hfq in vivo can be reconciled with biochemically stable and very slowly dissociating Hfq-RNA complexes is the topic of this review. Several recent reports suggest that the time scale discrepancy can be resolved by an active cycling model: rapid exchange of RNAs on Hfq is not limited by slow intrinsic dissociation rates, but is driven by the concentration of free RNA. Thus, transient binding of competitor RNA to Hfq-RNA complexes increases cycling rates and solves the strong binding/high turnover paradox.

  • 50.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Kill the messenger: bacterial antisense RNA promotes mRNA decay.2009Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 16, nr 8, s. 804-806Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial antisense RNAs target translation initiation regions (TIR s) to compete with ribosome binding, thus repressing translation and—secondarily—causing degradation of the naked mRNA. A new study reports on an antisense RNA that directly accelerates mRNA decay by targeting a sequence deep within the coding region, far downstream of the TIR.

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