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Identification of RNA molecules associated with the RNA-chaperone Hfq

ABSTRACT: We used high-throughput methods to discover the Salmonella RNAs that are targeted by the bacterial Sm-like protein, Hfq. Our generic approach, using chromosomal epitope tagging and coIP-on-chip will also be useful to identify the post-transcriptional regulons of many other RNA-binding proteins. Keywords: coIP-chip

ORGANISM(S): Salmonella enterica Salmonella enterica subsp. enterica serovar Typhimurium

PROVIDER: GSE10149 | GEO | 2008/08/23

SECONDARY ACCESSION(S): PRJNA108221

REPOSITORIES: GEO

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Identification of RNA molecules associated with the RNA-chaperone Hfq

Project description:We used high-throughput methods to discover the Salmonella RNAs that are targeted by the bacterial Sm-like protein, Hfq. Our generic approach, using chromosomal epitope tagging and coIP-on-chip will also be useful to identify the post-transcriptional regulons of many other RNA-binding proteins. Keywords: coIP-chip To identify the direct Hfq RNA targets, we co-immunoprecipitated RNA from a strain expressing a 3xFLAG-tagged Hfq-protein using an anti-3xFLAG antibody. As a negative control, we used the same antibody (anti-3xFLAG) to co-immunoprecipitate RNA from an isogenic strain expressing the non-tagged Hfq-protein. Two independent biological replicates were generated.

2010-06-20 | E-GEOD-10149 | biostudies-arrayexpress

Hfq LiRIP-seq in Salmonella

Project description:We have developed a novel approach named LiRIP-seq to profile the global RNA-RNA interactome in Salmonella enterica. By pulse expressing T4 RNA ligase from an inducible pBAD promoter, LiRIP-seq enables in vivo proximity ligation of Hfq-bound RNAs to their interaction partners. This is followed by enrichment of ligation products (RNA chimeras) using Hfq-coIP and subsequent RNA-seq analysis.

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Steady-state analysis of genes regulated by the E. coli RNA chaperone, Hfq

Project description:cDNA microarray analysis to identify genes regulated by the RNA chaperone, Hfq. Four experiments were performed: 1/ Hfq+ vs Hfq- strains. 269 significantly differentially regulated genes were identified by SAM (Statistical Analysis of Microarrays), of which 120 changed more than 1.5 fold (48 increased and 72 decreased in hfq-). Amongst other genes, these experiments identified significant regulation of the sigma E and sigma 32 regulons. However, only genes induced by sigma E were similarly induced in hfq-; 8 operons repressed by sigma E were not repressed in hfq-. 2/ wt vs delta rseA. RseA is the antisigma factor for sigmaE. This comparison results in elevated steady-state levels of sigma E, and confirmed induction and repression of target regulon members. 3/ hfq+ vs hfq+ rpoE overexpression. RpoE encoding sigma E was overexpressed in an hfq+ background, confirming normal regulation of the sigma E regulon. 4/ hfq+ vs hfq- rpoE overexpression. Sigma E was overexpressed in an hfq- background. This demonstrated that 8 operons normally repressed by sigma E require hfq for this repression. The simple conclusion is that sigma E regulates small RNAs that, together with Hfq, bind target mRNAs and results in their rapid degradation. This study is detailed in Guisbert et al 2007 (J Bacteriol, 189:1963-73) Keywords: Genetic modification

2007-01-01 | GSE6444 | GEO

Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Project description:Hfq-coIP overgrowth

| PRJNA169138 | ENA

Functional characterization of the RNA chaperone Hfq in opportunistic human pathogen Stenotrophomonas maltophilia

Project description:Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immuno-compromised hosts. We constructed an hfq deletion mutant (Delta-hfq) of S. maltophilia, and compared the behaviour of wild-type and Delta-hfq S. maltophilia cells in a variety of assays. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Delta-hfq strains showed that Hfq regulates expression of genes encoding flagellar and fimbrial components, transmembrane proteins, as well as enzymes involved in different metabolic pathways. Moreover, we analysed expression of several sRNAs identified by dRNA-seq in wild-type. The accumulation of two sRNAs was strongly reduced in the absence of Hfq.

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Functional characterization of the RNA chaperone Hfq in opportunistic human pathogen Stenotrophomonas maltophilia

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RyhB-mRNA pairing in absence of the RNA chaperone Hfq in vivo

Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.

2021-09-22 | GSE113591 | GEO

RybB-mRNA pairing in absence of the RNA chaperone Hfq in vivo

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DsrA-mRNA pairing in absence of the RNA chaperone Hfq in vivo

2021-09-22 | GSE113587 | GEO

PAO1 Hfq-V RNA-seq

Project description:A key challenge for understanding the role(s) played by short, non-coding RNAs (sRNAs) in bacteria is identifying the mRNA targets regulated by the sRNAs. Because the Hfq protein mediates the interactions between many sRNAs and the corresponding target mRNAs, one apporach to identify the mRNA targets of sRNAs is to capture sRNA:mRNA interactions occuring on Hfq by exposing cells to UV-irradiation, which forms cross-links between nucleic acids and proteins. We subjected cells of P. aeruginosa strain PAO1 and a derivative of PAO1 harboring a C-terminal VSV-G epitope on Hfq to UV-irradiation, immune-precipitated the Hfq-RNA complexes, ligated neighboring RNA molecules together with RNA Ligase, and then purififed the resulting RNAs. These RNAs were converted into cDNA libraries and sequenced using the Illumina NextSeq platform and then subjected to RIL-seq analysis pipeline (version 0.78) to identify chimeric RNA molecules. We also performed RNA-seq for PAO1 ∆phrS cells harboring an empty vector (pEV) or a vector expressing PhrS (pPhrS).

2023-05-03 | GSE216134 | GEO

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