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

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.

SUBMITTER: Sittka A

PROVIDER: S-EPMC2515195 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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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:BACKGROUND:Yersinia pestis is the causative agent of plague, which is transmitted primarily between fleas and mammals and is spread to humans through the bite of an infected flea or contact with afflicted animals. Hfq is proposed to be a global post-transcriptional regulator that acts by mediating interactions between many regulatory small RNAs (sRNAs) and their mRNA targets. Sequence comparisons revealed that Y. pestis appears to produce a functional homologue of E. coli Hfq. METHODOLOGY AND PRINCIPAL FINDINGS:Phenotype comparisons using in vitro assays demonstrated that Y. pestis Hfq was involved in resistance to H(2)O(2), heat and polymyxin B and contributed to growth under nutrient-limiting conditions. The role of Hfq in Y. pestis virulence was also assessed using macrophage and mouse infection models, and the gene expression affected by Hfq was determined using microarray-based transcriptome and real time PCR analysis. The macrophage infection assay showed that the Y. pestis hfq deletion strain did not have any significant difference in its ability to associate with J774A.1 macrophage cells. However, hfq deletion appeared to significantly impair the ability of Y. pestis to resist phagocytosis and survive within macrophages at the initial stage of infection. Furthermore, the hfq deletion strain was highly attenuated in mice after subcutaneous or intravenous injection. Transcriptome analysis supported the results concerning the attenuated phenotype of the hfq mutant and showed that the deletion of the hfq gene resulted in significant alterations in mRNA abundance of 243 genes in more than 13 functional classes, about 23% of which are known or hypothesized to be involved in stress resistance and virulence. CONCLUSIONS AND SIGNIFICANCE:Our results indicate that Hfq is a key regulator involved in Y. pestis stress resistance, intracellular survival and pathogenesis. It appears that Hfq acts by controlling the expression of many virulence- and stress-associated genes, probably in conjunction with small noncoding RNAs.

Dataset Information

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

Publications

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

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