| 1. |
- Stenum, Thomas Søndergaard, et al.
(författare)
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RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:9, s. 4572-4587
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 11
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Babina, Arianne M., et al.
(författare)
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Rescue of Escherichia coli auxotrophy by de novo small proteins
- 2023
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Berggren, Sofia, et al.
(författare)
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ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
- 2024
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Ingår i: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Holmqvist, Diana, 1984-
(författare)
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Adult Education at Auction : On Tendering-Based Procurement and Valuation in Swedish Municipal Adult Education
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 6. |
- Holmqvist, Diana, 1984-, et al.
(författare)
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Auctioning out education : On exogenous privatisation through public procurement
- 2021
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Ingår i: European Educational Research Journal. - : SAGE Publications. - 1474-9041. ; 20:1, s. 102-117
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Holmqvist, Erik, 1977-, et al.
(författare)
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RNA-binding activity and regulatory functions of the emerging sRNA-binding protein ProQ
- 2020
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Ingår i: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms. - : Elsevier BV. - 1874-9399 .- 1876-4320. ; 1863:9
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Forskningsöversikt (refereegranskat)abstract
- Regulatory small RNAs (sRNAs) ubiquitously impact bacterial physiology through antisense-mediated control of mRNA translation and stability. In Gram negative bacteria, sRNAs often associate with RNA-binding proteins (RBPs), both to gain cellular stability and to enable regulatory efficiency. The Hfq and CsrA proteins were for long the only known global RBPs implicated in sRNA biology. During the last five years, the FinO domain-containing protein ProQ has emerged as another global RBP with a broad spectrum of sRNA and mRNA ligands. This review provides a summary of the current knowledge of enterobacterial ProQ, with a special focus on RNA binding activity, RNA ligand preferences, influence on RNA stability and gene expression, and impact on bacterial physiology. Considering that characterization of ProQ is still in its infancy, we highlight aspects that, when addressed, will provide important clues to the physiological functions and regulatory mechanisms of this globally acting RBP.
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| 8. |
- Rizvanovic, Alisa, et al.
(författare)
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Saturation mutagenesis charts the functional landscape of Salmonella ProQ and reveals a gene regulatory function of its C-terminal domain
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:17, s. 9992-10006
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Tidskriftsartikel (refereegranskat)abstract
- The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.
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| 9. |
- Rizvanovic, Alisa, et al.
(författare)
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The RNA-binding protein ProQ promotes antibiotic persistence in Salmonella
- 2022
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial populations can survive the exposure to antibiotics through transient phenotypic and gene expression changes. These changes can be attributed to a small subpopulation of bacteria, giving rise to antibiotic persistence. Even though this phenomenon has been known for decades, much is still to be learnt about the mechanisms that drive persister formation. The RNA-binding protein ProQ has recently emerged as a global regulator of gene expression. Here, we show that ProQ impacts persister formation in Salmonella. ProQ contributes to growth-arrest in single cells, which are able to survive treatment with high concentrations of different antibiotics. The underlying mechanism for ProQ-dependent persister formation involves activation of the flagellar pathway. Importantly, we show that the ProQ-dependent phenotype is relevant during macrophage infection and allows Salmonella to survive the combined action of host immune defences and antibiotics. Together, our data highlights the importance of ProQ in Salmonella persistence and pathogenesis.
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| 10. |
- Romilly, Cedric, et al.
(författare)
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Small RNAs OmrA and OmrB promote class III flagellar gene expression by inhibiting the synthesis of anti-Sigma factor FlgM
- 2020
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 17:6, s. 872-880
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria can move by a variety of mechanisms, the best understood being flagella-mediated motility. Flagellar genes are organized in a three-tiered cascade allowing for temporally regulated expression that involves both transcriptional and post-transcriptional control. The class I operon encodes the master regulator FlhDC that drives class II gene transcription. Class II genes include fliA and flgM, which encode the Sigma factor sigma(28), required for class III transcription, and the anti-Sigma factor FlgM, which inhibits sigma(28) activity, respectively. The flhDC mRNA is regulated by several small regulatory RNAs (sRNAs). Two of these, the sequence-related OmrA and OmrB RNAs, inhibit FlhD synthesis. Here, we report on a second layer of sRNA-mediated control downstream of FhlDC in the flagella pathway. By mutational analysis, we confirm that a predicted interaction between the conserved 5MODIFIER LETTER PRIME seed sequences of OmrA/B and the early coding sequence in flgM mRNA reduces FlgM expression. Regulation is dependent on the global RNA-binding protein Hfq. In vitro experiments support a canonical mechanism: binding of OmrA/B prevents ribosome loading and decreases FlgM protein synthesis. Simultaneous inhibition of both FlhD and FlgM synthesis by OmrA/B complicated an assessment of how regulation of FlgM alone impacts class III gene transcription. Using a combinatorial mutation strategy, we were able to uncouple these two targets and demonstrate that OmrA/B-dependent inhibition of FlgM synthesis liberates sigma(28) to ultimately promote higher expression of the class III flagellin gene fliC.
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| 11. |
- Søndergaard Stenum, Thomas, et al.
(författare)
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CsrA enters Hfq's territory : Regulation of a base-pairing small RNA
- 2022
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 117:1, s. 4-9
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Post-transcriptional regulatory networks in Gammaproteobacteria are to a large extent built around the two globally acting RNA-binding proteins (RBPs) CsrA and Hfq. Both RBPs interact with small regulatory RNAs (sRNAs), but the functional outcomes of these interactions are generally distinct. Whereas Hfq both stabilizes sRNAs and promotes their base-pairing to target mRNAs, the sRNAs bound by CsrA act as sequestering molecules that titrate the RBP away from its mRNA targets. In this issue of Molecular Microbiology, Lai et al. reveal that CsrA interacts with the Hfq-associated and base-pairing sRNA Spot 42. In this case, CsrA increases Spot 42 stability by masking a cleavage site for endoribonuclease RNase E, thereby promoting Spot 42-dependent regulation of srlA mRNA. Interestingly, the effect of CsrA on srlA expression is two-fold. In addition to affecting Spot 42-dependent regulation, CsrA directly inhibits translation of SrlM, an activator of srlA transcription. Together, this study reveals a new function for CsrA and indicates more intricate connections between the CsrA and Hfq networks than previously anticipated. Several recent studies have identified additional RBPs that interact with sRNAs. With new RBP identification methods at hand, it will be intriguing to see how many more sRNA-binding proteins will be uncovered.
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| 12. |
- Wagner, E. Gerhart H., et al.
(författare)
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The Length of a DNA T-Tract Modulates Expression of a Virulence-Regulating sRNA
- 2020
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 80:2, s. 175-177
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Eisenbart et al. (2020) find an SSR-associated sRNA, NikS, that is subject to variable repeat-controlled expression. NikS regulates H. pylori virulence by post-transcriptionally repressing pathogenicity factors, including CagA and VacA, via base-pairing to their mRNAs.
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