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Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.ORCID iD: 0000-0001-7808-7781
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.ORCID iD: 0000-0001-7834-1487
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.ORCID iD: 0000-0003-2771-0486
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.

Place, publisher, year, edition, pages
2019. article id e101199
Keywords [en]
sRNA, Hfq, OmrA, OmrB, YdaM, Biofilm
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-343525DOI: 10.15252/embj.2018101199ISI: 000462892600006PubMedID: 30833291OAI: oai:DiVA.org:uu-343525DiVA, id: diva2:1186271
Funder
Swedish Research CouncilAvailable from: 2018-02-28 Created: 2018-02-28 Last updated: 2019-05-03Bibliographically approved
In thesis
1. Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
Open this publication in new window or tab >>Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is nowadays widely accepted that non-coding RNAs play important roles in post-transcriptional regulation of genes in all kingdoms of life. In bacteria, the largest group of RNA regulators are the small RNAs (sRNAs). Almost all sRNAs act through anti-sense base-pairing with target mRNAs, and by doing so regulate their translation and/or stability. As important modulators of gene expression, sRNAs are involved in all aspects of bacterial physiology. My studies aimed to deepen our understanding of the mechanisms behind sRNA-mediated gene regulation. We have shown that translation of the di-guanylate-cyclase YdaM, a major player in the biofilm regulatory cascade, is repressed by the sRNAs OmrA and OmrB. OmrAB require the RNA chaperone protein Hfq for efficient regulation. Interestingly, our results suggest a non-canonical mechanism for Hfq-mediated ydaM-OmrA/B base-pairing. Instead of serving as RNA interaction platform, Hfq restructures the ydaM mRNA to enable sRNA binding. We also addressed the question of how bacteria utilize regulatory RNAs to create phenotypic heterogeneity by studying the role of the tisB/istR-1 type 1 toxin-antitoxin system in SOS-induced persister cell formation in E. coli.

In addition, I have investigated the prokaryotic CRISPR-Cas immune system, which has led to the development of two molecular tools. The CRISPR-Cas adaptive immune system consists of a CRISPR array, where palindromic repeats are interspaced by unique spacer sequences derived from foreign genetic elements, and the CRISPR-associated (Cas) proteins. In the adaptation stage, memory is created by insertion of spacer sequences into the CRISPR array. We developed a fluorescent reporter that accurately and sensitively detects spacer integration events (denoted: “acquisition”) in single cells and in real-time. In the effector stage of immunity, crRNAs, consisting of one spacer-repeat unit, associate with the Cas proteins to form a ribonucleoprotein complex that surveys the cell for invader DNA. Target identification occurs by base-pairing between the crRNA and the complementary sequence in the target nucleic acid, which triggers degradation. We repurposed the E. coli type I-E CRISPR-Cas effector complex Cascade for specific reprogrammable transcriptional gene silencing.

The studies presented herein thus contributes to our understanding of RNA-based target identification for gene regulation and adaptive immunity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 83
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1637
Keywords
small RNA, sRNA, non-coding RNA, Hfq, gene regulation, post-transcriptional regulation, biofilm, OmrA, OmrB, toxin-antitoxin, TisB, IstR-1, Persisters, CRISPR-Cas, CRISPR-Cas adaptation reporter, Escherichia coli
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-343526 (URN)978-91-513-0252-2 (ISBN)
Public defence
2018-04-20, A1:111a, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2018-03-26 Created: 2018-02-28 Last updated: 2018-04-24

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Hoekzema, MirtheRomilly, CedricHolmqvist, ErikWagner, Gerhart E. H.

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