Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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 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.

ORGANISM(S): Salmonella enterica

SUBMITTER: Cynthia Sharma

PROVIDER: E-GEOD-10149 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq.

Sittka Alexandra A Lucchini Sacha S Papenfort Kai K Sharma Cynthia M CM Rolle Katarzyna K Binnewies Tim T TT Hinton Jay C D JC Vogel Jörg J

PLoS genetics 20080822 8

Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigm ...[more]

PMID: 18725932

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Project description:The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial âcoreâ small RNA of unknown function. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of >16% (â¥750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs suggesting that the unprecedented degree of pleiotropic regulation might in part be caused by titration of Hfq binding by ArcZ. This study used three different approaches to identify target genes and biological role of the small RNA ArcZ in Salmonella Typhimurium. Transcriptomic analysis of ArcZ overexpression: Strain JVS-0082 (ÎarcZ) was transformed with plasmids pJV300 (control) and pKP48-1 (parcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptomic analysis of arcZ mutant strain: Strains JVS-007 (WT) and JVS-0082 (ÎarcZ) were transformed with plasmid pJV300 (control) and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptional effects of ArcZ pulse expression: Strain SL1344 was transformed with plasmids pKP8-35 (pBAD-control) and pKP4-13 (pBAD-ArcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture to an OD600 of 1.5. Expression of the insert was induced with L-arabinose (0.2% final concentrations) for 10 min. Three biological replicates were performed for each strain/condition.

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Dataset Information

Identification of RNA molecules associated with the RNA-chaperone Hfq

Publications

Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq.

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