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BETA
Wagner, Gerhart E. H.ORCID iD iconorcid.org/0000-0003-2771-0486
Alternative names
Publications (10 of 59) Show all publications
Bass, B. L., O'Connell, M. A. & Wagner, G. E. H. (2019). Biographical item: "A celebration of the life of Marie Öhman (1964-2019)" in RNA, Volume: 25 Issue: 10, Pages: IX-XI. , 25(10)
Open this publication in new window or tab >>Biographical item: "A celebration of the life of Marie Öhman (1964-2019)" in RNA, Volume: 25 Issue: 10, Pages: IX-XI
2019 (English)Other (Other (popular science, discussion, etc.))
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-395622 (URN)10.1261/rna.072645.119 (DOI)000486307400001 ()31311820 (PubMedID)
Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-10-22Bibliographically approved
Hoekzema, M., Romilly, C., Holmqvist, E. & Wagner, G. E. H. (2019). Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation. EMBO Journal, Article ID e101199.
Open this publication in new window or tab >>Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
2019 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e101199Article in journal (Refereed) Published
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.

Keywords
sRNA, Hfq, OmrA, OmrB, YdaM, Biofilm
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-343525 (URN)10.15252/embj.2018101199 (DOI)000462892600006 ()30833291 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2019-05-03Bibliographically approved
Romilly, C., Deindl, S. & Wagner, G. E. H. (2019). The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element. Proceedings of the National Academy of Sciences of the United States of America, 116(32), 15901-15906
Open this publication in new window or tab >>The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element
2019 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 32, p. 15901-15906Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES, 2019
Keywords
translation initiation, ribosome standby site, RNA secondary structure, ribosomal protein S1, fluorescence anisotropy
National Category
Microbiology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-392132 (URN)10.1073/pnas.1904309116 (DOI)000478971900029 ()31320593 (PubMedID)
Funder
Swedish Research CouncilEU, European Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-03Bibliographically approved
Sterk, M., Romilly, C. & Wagner, G. E. H. (2018). Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.. Nucleic Acids Research, 46(8), 4188-4199
Open this publication in new window or tab >>Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 8, p. 4188-4199Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Oxford University Press, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-343078 (URN)10.1093/nar/gky073 (DOI)000431895800039 ()29420821 (PubMedID)
Funder
Swedish Research Council, VR 621-2010-5233
Available from: 2018-02-25 Created: 2018-02-25 Last updated: 2018-08-10Bibliographically approved
Amlinger, L., Hoekzema, M., Wagner, G. E. H., Koskiniemi, S. & Lundgren, M. (2017). Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition. Scientific Reports, 7, Article ID 10392.
Open this publication in new window or tab >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
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.

Keywords
quorum sensing controls, escherichia-coli, cas adaptation, immune-system, host factor, dna, resistance, elements, repeats, bacteriophage
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-334399 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()
Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Amlinger, L., Hoekzema, M., Wagner, G. E. H., Koskiniemi, S. & Lundgren, M. (2017). Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition. Scientific Reports, 7, Article ID 10392.
Open this publication in new window or tab >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-335401 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()28871175 (PubMedID)
Funder
Swedish Research CouncilWenner-Gren FoundationsThe Royal Swedish Academy of SciencesScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2017-12-05 Created: 2017-12-05 Last updated: 2018-02-28Bibliographically approved
Holmqvist, E. & Wagner, E. G. (2017). Impact of bacterial sRNAs in stress responses. Biochemical Society Transactions, 45, 1203-1212
Open this publication in new window or tab >>Impact of bacterial sRNAs in stress responses
2017 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 45, p. 1203-1212Article, review/survey (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-347714 (URN)10.1042/BST20160363 (DOI)000418440200002 ()29101308 (PubMedID)
Funder
Swedish Research CouncilWenner-Gren FoundationsSwedish Foundation for Strategic Research
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2018-04-06Bibliographically approved
Berghoff, B. A. & Wagner, G. E. H. (2017). RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence. Current Genetics, 63(6), 1011-1016
Open this publication in new window or tab >>RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence
2017 (English)In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 63, no 6, p. 1011-1016Article, review/survey (Refereed) Published
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.

Keywords
Toxin-antitoxin, Antisense RNA, 5 ' UTR structure, Persistence, Depolarization, SOS response
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-346606 (URN)10.1007/s00294-017-0710-y (DOI)000414500800010 ()28560584 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2018-03-22Bibliographically approved
Berghoff, B. A., Karlsson, T., Kallman, T., Wagner, G. E. H. & Grabherr, M. G. (2017). RNA-sequence data normalization through in silico prediction of reference genes: the bacterial response to DNA damage as case study. BioData Mining, 10, Article ID 30.
Open this publication in new window or tab >>RNA-sequence data normalization through in silico prediction of reference genes: the bacterial response to DNA damage as case study
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2017 (English)In: BioData Mining, ISSN 1756-0381, E-ISSN 1756-0381, Vol. 10, article id 30Article in journal (Refereed) Published
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.

Keywords
RNA-seq, Transcriptomics, Normalization, Gene expression, DNA damage, Stress response
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-334397 (URN)10.1186/s13040-017-0150-8 (DOI)000409118300001 ()28878825 (PubMedID)
Funder
Swedish Research Council FormasSwedish Research Council, 621-2010-5233
Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2019-11-20Bibliographically approved
Berghoff, B. A., Hoekzema, M., Aulbach, L. & Wagner, G. E. H. (2017). Two regulatory RNA elements affect TisB-dependent depolarization and persister formation. Molecular Microbiology, 103(6), 1020-1033
Open this publication in new window or tab >>Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
2017 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 6, p. 1020-1033Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2017
National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
urn:nbn:se:uu:diva-320870 (URN)10.1111/mmi.13607 (DOI)000395758900008 ()27997707 (PubMedID)
Funder
German Research Foundation (DFG), BE 5210/1-1 BE 5210/2-1Swedish Research Council, VR 621-2010-5233
Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2018-02-28Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-2771-0486

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