Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3′ Ends

Holmqvist, Erik; Li, Lei; Bischler, Thorsten; Barquist, Lars; Vogel, Joerg

doi:10.1016/j.molcel.2018.04.017

Cited by 132 publications

(288 citation statements)

References 88 publications

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“…We wondered whether the ProQ-cspE B3H interaction might be limited by competition with endogenous ProQ or cellular Hfq, given that co-immunoprecipitation studies have suggested that ProQ and Hfq may compete for a subset of their RNA substrates. (22,37) While no additional stimulation of transcription over basal levels was observed when the ProQ-cspE B3H experiment was repeated in ΔproQ reporter cells ( Fig 1C; 1.2), we found that the fold-stimulation of β-gal transcription indeed increased in Δhfq reporter cells ( Fig 1D; 2.3x). This Δhfq reporter strain was previously used for detecting Hfq-RNA interactions, (30) and was used throughout the remainder of this study.…”

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 66%

“…Residues 2-119, 2-131, 2-176, 181-232 and 2-232 of E. coli ProQ were fused to the ɑ-NTD (residues 1-248) in pBRɑ between NotI and BamHI to generate pSP90 (pBRɑ-ProQ NTD ), pKB951 (pBRɑ-ProQ NTD+12aa ), pKB955 (pBRɑ-ProQ ΔCTD ), pSP92 (pBRɑ-ProQ CTD ) and pKB949 (pBRɑ-ProQ FL , full-length) respectively. pCW17 (pAC-p constit -λCI-MS2 CP ) was derived from pKB989 (pAC-p lacUV5 -λCI-MS2 CP ) (22) by substitution of the region between -35 and +22 of the p lacUV5 promoter (containing the -35, -10 and lacO elements) with the following sequence lacking a lacO element (predicted -35, -10 and TSS of the resulting constitutive promoter are underlined):…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…The cspE 3'UTR was chosen as an initial RNA candidate due to strong interaction with ProQ that has been observed both in vivo and in vitro. (22,28) To simplify the possibility of multiple ProQ domains interacting with RNA, we began our analysis with a construct possessing only the ProQ NTD and linker (resi 2-176; hereafter ProQ ΔCTD ). For the envisioned assay, ProQ ΔCTD , the "prey" protein, is fused to the N-terminal domain of the alpha subunit of RNAP (α; Fig 1A).…”

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

“…Given that ProQ has been found to interact with dozens of sRNAs and hundreds of mRNAs inside of Salmonella and E. coli cells, (17) we sought to determine whether our B3H system could detect ProQ interactions with additional RNAs beyond cspE. We tested three additional RNAs that had been found to interact in vivo with ProQ: sRNAs SibB, RyjB and the 3'UTR of fbaA (hereafter, fbaA); (17,22,37) as well as four sRNAs for which we had previously detected B3H interactions with Hfq: ChiX, OxyS, ArcZ, and MgrR ( Fig S3). (30) While a cspE hybrid RNA consistently produced the highest stimulation of transcription above basal levels when present with α-ProQ Δ CTD , we observed a ≥1.5-fold increase in fold-stimulation of β-gal activity when each of these hybrid RNAs was present in reporter cells (Fig 2A; full b-gal data in Fig S4A).…”

Section: Scope Of Detectable Rna Interactionsmentioning

confidence: 99%

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Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Pandey

Gravel

Stockert

et al. 2019

Preprint

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The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs.Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex. INTRODUCTION Regulatory, small RNAs (sRNAs) are found in nearly all bacterial species and implicated in important processes such as virulence, biofilm formation, host interactions and antibiotic resistance.(1-3) These sRNAs typically regulate messenger RNA (mRNA) translation through imperfect base pairing near an mRNA's ribosomal binding site.(2, 4-6) In many bacterial species, the stability and function of sRNAs are supported by global RNA-binding proteins, such as the protein Hfq.(1, 4, 7-9) Given that Hfq is not present in all bacterial species and that not all sRNAs depend on Hfq for their function, there is increasing interest in other RNA-binding proteins that may play a role in global gene-regulation in bacteria,(2, 10-13) including a class of proteins that contain FinO domains.(14-17) The Escherichia coli protein FinO is the founding member of the FinO structural class of RNA-binding proteins. In E. coli, FinO binds the FinP sRNA and regulates the 5´ untranslated region (UTR) of traJ.(18, 19) Similarly, Legionella pneumophila RocC contains a FinO-domain and binds the sRNA RocR along with at least four 5' UTRs of mRNAs involved in competence.(20) In E. coli, another FinO-domain-containing protein called ProQ was initially characterized as an RNA-binding protein contributing to osmoregulation through expression of proP.(21) ProQ was recently identified through Grad-Seq experiments to bind to dozens of cellular RNAs,(17) including a large number of sRNAs and mRNA 3'UTRs in Samonella and E. coli.(22) ProQ binding has been shown to regulate mRNA-expression levels through interactions with both 5' and 3' UTRs. It has been shown to form a ternary complex with an sRNA (RaiZ) and an mRNA (hupA), to support RaiZ's repression of hupA,(23) and to protect mRNAs from exonucleolytic degradation by binding to 3' ends.(22) Further, ProQ supports the sRNA SraL in preventin...

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Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 66%

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

Section: Scope Of Detectable Rna Interactionsmentioning

confidence: 99%

See 2 more Smart Citations

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Pandey

Gravel

Stockert

et al. 2019

Preprint

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“…For low aeration conditions, overnight NSLB cultures were subcultured 1/100 into 16 ml of HSLB in a 20 × 150 mm tube and incubated for 3 h on a platform shaker at 225 rpm. For the measurement of the half‐life of hilD mRNA, 500 μg of rifampicin was added into the bacterial cultures (Holmqvist et al , ). Immediately upon addition of rifampicin (time 0) and at 1, 2, 4, 8 and 16 min after the treatment, 800 μl aliquots of bacterial cells were collected.…”

Section: Methodsmentioning

confidence: 99%

Oxygen‐dependent regulation of SPI1 type three secretion system by small RNAs in Salmonella enterica serovar Typhimurium

Kim

Golubeva

Vanderpool

et al. 2018

Molecular Microbiology

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Summary Salmonella Typhimurium induces inflammatory diarrhea and uptake into intestinal epithelial cells using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Three AraC‐like regulators, HilD, HilC and RtsA, form a feed‐forward regulatory loop that activates transcription of hilA, encoding the activator of the T3SS structural genes. Many environmental signals and regulatory systems are integrated into this circuit to precisely regulate SPI1 expression. A subset of these regulatory factors affects translation of hilD, but the mechanisms are poorly understood. Here, we identified two sRNAs, FnrS and ArcZ, which repress hilD translation, leading to decreased production of HilA. FnrS and ArcZ are oppositely regulated in response to oxygen, one of the key environmental signals affecting expression of SPI1. Mutational analysis demonstrates that FnrS and ArcZ bind to the hilD mRNA 5′ UTR, resulting in translational repression. Deletion of fnrS led to increased HilD production under low‐aeration conditions, whereas deletion of arcZ abolished the regulatory effect on hilD translation aerobically. The fnrS arcZ double mutant has phenotypes in a mouse oral infection model consistent with increased expression of SPI1. Together, these results suggest that coordinated regulation by these two sRNAs maximizes HilD production at an intermediate level of oxygen.

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Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products

Evguenieva‐Hackenberg

2021

WIREs RNA

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Gene expression strategies ensuring bacterial survival and competitiveness rely on cis‐ and trans‐acting RNA‐regulators (riboregulators). Among the cis‐acting riboregulators are transcriptional and translational attenuators, and antisense RNAs (asRNAs). The trans‐acting riboregulators are small RNAs (sRNAs) that bind proteins or base pairs with other RNAs. This classification is artificial since some regulatory RNAs act both in cis and in trans, or function in addition as small mRNAs. A prominent example is the archetypical, ribosome‐dependent attenuator of tryptophan (Trp) biosynthesis genes. It responds by transcription attenuation to two signals, Trp availability and inhibition of translation, and gives rise to two trans‐acting products, the attenuator sRNA rnTrpL and the leader peptide peTrpL. In Escherichia coli, rnTrpL links Trp availability to initiation of chromosome replication and in Sinorhizobium meliloti, it coordinates regulation of split tryptophan biosynthesis operons. Furthermore, in S. meliloti, peTrpL is involved in mRNA destabilization in response to antibiotic exposure. It forms two types of asRNA‐containing, antibiotic‐dependent ribonucleoprotein complexes (ARNPs), one of them changing the target specificity of rnTrpL. The posttranscriptional role of peTrpL indicates two emerging paradigms: (1) sRNA reprograming by small molecules and (2) direct involvement of antibiotics in regulatory RNPs. They broaden our view on RNA‐based mechanisms and may inspire new approaches for studying, detecting, and using antibacterial compounds. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule‐RNA Interactions RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs

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Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3′ Ends

Cited by 132 publications

References 88 publications

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Oxygen‐dependent regulation of SPI1 type three secretion system by small RNAs in Salmonella enterica serovar Typhimurium

Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products

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