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Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons82165

Sittka,  Alexandra
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82089

Papenfort,  Kai
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82158

Sharma,  Cynthia Mira
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82123

Rolle,  Katarzyna
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82198

Vogel,  Jörg
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

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PLoS_Genetics_2008_4_e1000163.pdf
(Verlagsversion), 708KB

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Zitation

Sittka, A., Lucchini, S., Papenfort, K., Sharma, C. M., Rolle, K., Binnewies, T. T., et al. (2008). Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq. PLoS Genetics, 4(8): e1000163.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-000E-C191-5
Zusammenfassung
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.