| 1. |
- Stenum, Thomas Søndergaard, et al.
(author)
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RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins
- 2023
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:9, s. 4572-4587
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Journal article (peer-reviewed)abstract
- RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
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| 2. |
- Andresen, Liis, et al.
(author)
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CLIP-Seq in Bacteria : Global Recognition Patterns of Bacterial RNA-Binding Proteins
- 2018
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In: High-Density Sequencing Applications in Microbial Molecular Genetics. - : ELSEVIER ACADEMIC PRESS INC. - 9780128159934 ; , s. 127-145
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Book chapter (peer-reviewed)abstract
- RNA-protein interactions are at the heart of many central cellular processes, and RNA-binding proteins (RBPs) associate with virtually all RNA molecules in a cell. In bacteria, global RBPs, often in conjunction with small regulatory RNAs, affect physiology and virulence by controlling transcription, translation, and RNA decay. To understand how these regulatory proteins orchestrate global gene expression, detailed maps of their cellular RNA binding sites are required. To this end, cross-linking and immunoprecipitation followed by deep sequencing (CLIP-seq) has revolutionized RBP studies by providing knowledge about global recognition patterns of RBPs in both eukaryotic and bacterial cells. In this chapter, we provide a step-by-step protocol for global mapping of bona fide RBP binding sites using CLIP-seq in bacteria. This protocol has been successfully applied for charting the binding sites of Hfq, CsrA, and ProQ, three global regulatory RBPs in Salmonella enterica and Escherichia coli, and should be readily applicable to other RBPs and bacterial species.
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| 3. |
- Andresen, Liis, et al.
(author)
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The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR
- 2020
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In: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 11
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Journal article (peer-reviewed)abstract
- Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.
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| 4. |
- Babina, Arianne M., et al.
(author)
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Rescue of Escherichia coli auxotrophy by de novo small proteins
- 2023
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In: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12
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Journal article (peer-reviewed)abstract
- Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (<= 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5 ' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.
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| 5. |
- Berggren, Sofia, et al.
(author)
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ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
- 2024
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In: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
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Journal article (peer-reviewed)abstract
- Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3 ' UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
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| 6. |
- Eleftheraki, Athina, et al.
(author)
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An RNA pseudoknot mediates toxin translation and antitoxin inhibition
- 2024
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 121:27
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Journal article (peer-reviewed)abstract
- Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth- inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation- competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full- length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.
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| 7. |
- Gonzalez, Grecia M., et al.
(author)
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Structure of the Escherichia coli ProQ RNA-binding protein
- 2017
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In: RNA. - : COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. - 1355-8382 .- 1469-9001. ; 23:5, s. 696-711
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Journal article (peer-reviewed)abstract
- The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia call ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
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| 8. |
- Hoekzema, Mirthe, et al.
(author)
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Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
- 2019
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In: EMBO Journal. - : EMBO Press. - 0261-4189 .- 1460-2075. ; 38:7
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Journal article (peer-reviewed)abstract
- 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.
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| 9. |
- Holmqvist, Diana, 1984-
(author)
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Adult Education at Auction : On Tendering-Based Procurement and Valuation in Swedish Municipal Adult Education
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Our understanding of adult education’s purpose and value is linked to how we think ‘good’ education should be organised and provided. This thesis explores the interplay between organisation and values by looking at how conceptualisations and valuations of adult education are affected when private providers become involved in public education.For this purpose, the study draws on the example of Swedish municipal adult education (MAE). On the one hand, Swedish adult education is organised to compensate for structural inequalities and mitigate barriers to participation in education for citizens. On the other hand, private providers are allowed to make profits from their involvement in public education. Sweden’s combined commitment to universal welfare and the market make this a particularly interesting case to examine. Mainly contracted through tendering-based procurement, for-profit companies (and to a lesser extent non-public organisations) now service almost half of all MAE course participants. The thesis explores how municipalities construct the value and purpose of MAE when placing orders with private providers, and how teachers working in MAE reflect upon the purpose and value of MAE.Drawing on empirical data from both procurements and interviews, the study shows that municipalities use tendering-based procurement to shape the public-private partnership and promote different values. On a national level, this creates a heterogenous landscape of adult education provision and valuations. Furthermore, the study shows that there are tensions between MAE teachers’ professional self-conceptualisations and the realities put forth by policy or constructed in the procurement process. Analysing how teachers navigate these tensions, the study shows that teachers work to resist reformation, while they also become complicit in enforcing values promoted through policy and procurement.
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| 10. |
- Holmqvist, Diana, 1984-, et al.
(author)
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Auctioning out education : On exogenous privatisation through public procurement
- 2021
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In: European Educational Research Journal. - : SAGE Publications. - 1474-9041. ; 20:1, s. 102-117
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Journal article (peer-reviewed)abstract
- Privatisation of public education is becoming more and more common across the world. As much current research presupposes causal links between the degree of privatisation and issues of competition and student?s free choice, we see a need for research on other ways of organising the presence of private providers in public education. In this article, we study how two Swedish municipalities use public procurement to contract private providers and organise adult education. Interestingly, we find that competition is more heavily at play in the municipality that outsources half of its adult education, than in the municipality that outsources all adult education. We view these findings as vital for understanding how education is being outsourced to private providers and for furthering the discussion on the consequences of the ongoing privatisation of education.
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