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UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

ABSTRACT: UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

PROVIDER: PRJNA197291 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR828515.fastq.gz	Fastqsanger.gz
	SRR828516.fastq.gz	Fastqsanger.gz
	SRR828517.fastq.gz	Fastqsanger.gz
	SRR828518.fastq.gz	Fastqsanger.gz
	SRR828519.fastq.gz	Fastqsanger.gz

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UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

2014-05-22 | GSE46118 | GEO

UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

Project description:UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms. His-FLAG tagged Hfq and untagged controls were cultured to an OD of 0.8 and crosslinked with UV-C. Five replicates of tagged EHEC Hfq and 2 replicates of untagged Hfq were crosslinked. We have additionally crosslinked 2 replicates each of E. coli K12 tagged and untagged Hfq.

2014-05-22 | E-GEOD-46118 | biostudies-arrayexpress

Global RNA recognition patterns of post‐transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo

Project description:The molecular roles of many RNA‐binding proteins in bacterial post‐transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP‐seq) have begun to revolutionize the transcriptome‐wide mapping of eukaryotic RNA‐binding protein target sites. We have applied CLIP‐seq to chart the target landscape of two major bacterial post‐transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single‐nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3′‐located Rho‐independent terminators as a universal motif involved in Hfq–RNA interactions. Additionally, Hfq preferentially binds 5′ to sRNA‐target sites in mRNAs, and 3′ to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA–mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA‐target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP‐seq approach will bring new insights into post‐transcriptional gene regulation by RNA‐binding proteins in diverse bacterial species.

2016-05-25 | GSE74425 | GEO

Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RNA-Seq]

Project description:Bacterial small non-coding RNAs (sRNAs) play post-transcriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the P. aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5’ UTRs and sRNAs, and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that sRNAs association with Hfq is primarily a function of their expression levels, strongly supporting that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of post-transcriptional regulation in P. aeruginosa.

2019-11-15 | GSE136111 | GEO

Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RIP-Seq]

2019-11-15 | GSE136110 | GEO

The S. cerevisiae m6A reader Pho92 promotes timely meiotic recombination by controlling key methylated transcripts [CRAC]

Project description:We performed CRAC (UV-crosslinking and analysis of cDNA) to identify Ime4-dependent mRNA targets of Pho92 during meiosis at 5 hr in SPO.

2022-07-08 | GSE200088 | GEO

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

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.

2012-10-02 | GSE39705 | GEO

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

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

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