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          Institute: MPI für Infektionsbiologie     Collection: RNA Biology     Display Documents



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ID: 399799.0, MPI für Infektionsbiologie / RNA Biology
Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq
Authors:Sittka, Alexandra; Lucchini, Sacha; Papenfort, Kai; Sharma, Cynthia Mira; Rolle, Katarzyna; Binnewies, Tim T.; Hinton, Jay C. D.; Vogel, Jörg
Language:English
Date of Publication (YYYY-MM-DD):2008-08
Title of Journal:PLoS Genetics
Journal Abbrev.:PLoS Genet.
Volume:4
Issue / Number:8
Sequence Number of Article:e1000163
Copyright:All site content, except where otherwise noted, is licensed under a Creative Commons Attribution License.
Review Status:Peer-review
Audience:Experts Only
Abstract / Description: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 sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria.
External Publication Status:published
Document Type:Article
Communicated by:Hilmar Fünning
Affiliations:MPI für Infektionsbiologie/RNA Biology
External Affiliations:Inst Food Res, Inst Food Res, Norwich NR4 7UA, Norfolk, England.; Tech Univ Denmark, Ctr Biol Sequence Anal, DK-2800 Lyngby, Denmark.
Identifiers:ISI:000260410800013 [ID No:1]
ISSN:1553-7390 [ID No:2]
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