A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

Berry, Katie; Hochschild, Ann

doi:10.1093/nar/gkx1086

Cited by 15 publications

(42 citation statements)

References 59 publications

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“…A single-copy O L 2-62-lacZ reporter on an F' episome bearing tetracycline resistance was generated as previously described (31,32) by conjugative delivery of pFW11-derivative plasmid pFW11-O L 2-tet into FW102 cells to generate Escherichia coli strain KB480, which is analogous to O L 2-62-lacZ reporters carried on F' episomes bearing kanamycin resistance. (30,33) The Δhfq::kan allele and ΔproQ::kan allele from the Keio collection (34) were introduced to KB480 via P1 transduction to generate KB483 and SP2 respectively. An analogous process was used to create strain KB511 from by conjugative delivery of pFW11-derivative plasmid pKB1067 into FW102 cells.…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…Our previous studies with the bacterial three-hybrid (B3H) assay have focused on Hfq-RNA interactions. (30) We sought to determine whether this B3H assay could detect interactions of ProQ with RNA in an analogous manner to the interactions of RNA and Hfq. 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.…”

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

“…(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. To confirm the ProQ-cspE B3H interaction represented a specific interaction, we sought to disrupt it with a point substitution.…”

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

“…We have previously reported a transcription-based bacterial three-hybrid (B3H) assay that facilitates the detection of RNA-protein interactions inside of living E. coli reporter cells. (30) While this assay was effective in detecting numerous Hfq-sRNA interactions, it was unclear how generally applicable this assay would be to other RNA-protein interactions. Here we present a modified B3H assay that is able to robustly and specifically detect ProQ-RNA interactions, and provides a path to apply the tools of molecular genetics to the mechanism of ProQ-RNA interactions.…”

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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: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

Section: Establishing a B3h Assay For Proq-rna Interactionsmentioning

confidence: 99%

mentioning

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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“…An important question is whether these adenosines play a similar role in controlling Hfq-and ProQ-interactions in vivo. To investigate this, we utilized a bacterial three-hybrid (B3H) system for RNA-protein interactions that has been demonstrated to detect both Hfq-and ProQ-RNA interactions in vivo (30,51). In this assay, expression of three hybrid components make reporter-gene (lacZ) transcription from a test promoter dependent on a productive interaction between an RNA 'bait' and a protein 'prey' (Fig.…”

Section: In Vivo Rna-binding Determinants For Proq and Hfqmentioning

confidence: 99%

Determinants of RNA recognition by the FinO domain of theEscherichia coliProQ protein

Stein

Kwiatkowska

Basczok

et al. 2020

Preprint

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The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3ʹ-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least two base pairs preceding an 3ʹ oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5ʹ side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3ʹ and cspE-3ʹ RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3ʹ intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.RNA-binding proteins are important contributors to major life processes, including the regulation of gene expression by RNAs (1). In many bacteria, the prominent role played by trans-encoded base-pairing small RNAs (sRNAs) in regulating gene expression requires a matchmaker protein called Hfq, which assists sRNA in pairing to complementary sequences in target mRNAs and affects sRNA stability (2-6). Global identification of new RNA/protein interactions has been enabled by deep-sequencing approaches (7), such as Grad-seq, which uses glycerol gradients to identify novel RNA/protein complexes (8), CLIP-seq, which uses crosslinking to define protein binding sites in the transcriptome (9), and RIL-seq which identifies protein-dependent RNA-RNA interactions (10). Recent studies using these approaches showed that another protein, named ProQ, is a global RNA binding protein in Escherichia coli and Salmonella enterica (11-13), and is involved in sRNA interactions with other RNA molecules (12,14,15). The pool of RNA ligands of ProQ is mostly distinct from that of Hfq and contains more mRNAs than sRNAs (11,12).While ProQ was originally discovered in a search for genes that affect proline transport (16), this protein contributes to several physiological processes in E.coli and S. enterica (17)including DNA metabolism (13,14), bacterial virulence (15), and adaptation to osmotic stress (12) and resource limitation (18). ProQ is active as a monomer (19), and it belongs to the FinO-domain family with representatives in numerous γ-proteobacteria (17,20). Other members of this family include the F-like plasmid FinO protein (21,22), Legionella pneumophila RocC protein (23), and S. enterica pCol1B9 plasmid-encoded FopA protein (J.Vogel, personal communication), which each interact with few RNAs...

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The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA–Protein Interactions in Living Mammalian Cells

et al. 2020

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RNA−protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA−protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein−protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA−protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA−protein interaction screening tool.

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A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

Cited by 15 publications

References 59 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

Determinants of RNA recognition by the FinO domain of theEscherichia coliProQ protein

The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA–Protein Interactions in Living Mammalian Cells

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