PMID:18725932
| Citation |
Sittka, A, Lucchini, S, Papenfort, K, Sharma, CM, Rolle, K, Binnewies, TT, Hinton, JC and Vogel, J (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet. 4:e1000163 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2515195 Online version:10.1371/journal.pgen.1000163 |
| Keywords |
Flagella/metabolism; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Host Factor 1 Protein/genetics; Host Factor 1 Protein/metabolism; Molecular Sequence Data; RNA Processing, Post-Transcriptional; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA, Untranslated/genetics; RNA, Untranslated/metabolism; Regulon; Salmonella typhimurium/genetics; Salmonella typhimurium/growth & development; Salmonella typhimurium/metabolism; Sequence Analysis, DNA/methods; Virulence |
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