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

Hfq. PLoS Genet 4(8): e1000163. doi:10.1371/journal.pgen.1000163 Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq Alexandra Sittka 0 Sacha Lucchini 0 Kai Papenfort 0 Cynthia M. Sharma 0 Katarzyna Rolle 0 Tim T. 0 Binnewies 0 Jay C. D. Hinton 0 Jo rg Vogel 0 William F. Burkholder, Stanford University, United States of America 0 1 Max Planck Institute for Infection Biology, RNA Biology, Berlin, Germany, 2 Institute of Food Research , Norwich Research Park, Norwich , United Kingdom , 3 Center for Biological Sequence Analysis, Technical University of Denmark , Lyngby , Denmark 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. - Funding: This work was supported by the core strategic grant of the BBSRC to the Hinton lab, and funds from the DFG Priority Program SPP1258 Sensory and Regulatory RNAs in Prokaryotes to the Vogel lab. Competing Interests: The authors have declared that no competing interests exist. Until now, global gene expression control studies have generally focussed on the transcriptional regulation exerted by the specific action of DNA binding proteins, and on the post-translational regulation governed by specific proteinprotein interactions. In comparison, little is known about how RNA binding proteins facilitate the global control of gene expression at the posttranscriptional level. However, the latest discoveries of many small noncoding RNAs (sRNAs) in both pro- and eukaryotes have shown that the interaction of RNA with proteins plays a prominent role in the regulation of cellular processes. In bacteria, the majority of the sRNAs basepair with target mRNAs to regulate their translation and/or decay [1,2,3], and these regulatory events commonly require the bacterial Sm-like protein, Hfq [4,5]. Hfq is one of the most abundant RNA-binding proteins in bacteria [6,7,8]. First identified in Escherichia coli as a host factor required for phage Qb RNA replication ,40 years ago [9], Hfq is now known to have an important physiological role in numerous model bacteria [5]. Almost half of all sequenced Gram-negative and Gram-positive species, and at least one archaeon, encode an Hfq homologue [10,11]. Hfq interacts with regulatory sRNAs and mRNAs, and much of its post-transcriptional function is caused by the facilitation of the generally short and imperfect antisense interactions of sRNAs and their targets [12,13,14,15,16,17]. However, Hfq can also act alone as a translational repressor of mRNA [18,19], and can modulate mRNA decay by stimulating polyadenylation [20,21]. In addition, roles of Hfq in tRNA biogenesis have recently been described [22,23]. The pleiotropy of an hfq deletion mutation was first apparent from the multiple stress response-related phenotypes in E. coli [24], and partly reflects the reduced efficiency of translation of rpoS mRNA, encoding the major stress sigma factor, sS [25,26]. However, Hfq clearly impacts on bacterial physiology in a much broader fashion, including the sS-independent control of virulence factors in pathogenic bacteria (e.g., [27,28,29,30, 31,32,33]). Specifically, deletion of hfq attenuates the ability of the model pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) to infect mice, to invade epithelial cells, to secrete virulence factors and to survive inside cultured macrophages [32]. The past decade has seen small regulatory RNA become an important new mediator of bacterial mRNA regulation. This study describes a rapid way to identify novel sRNAs that are expressed, and should prove relevant to a variety of bacteria. We purified the epitope-tagged RNA-binding protein, Hfq, and its bound RNA by immunoprecipitation from the model pathogen, Salmonella enterica serovar Typhimurium. This new strategy used Next Generation pyrosequencing to identify 727 Hfq-bound mRNAs. The numbers of sRNAs expressed in Salmonella was doubled to 64; half are associated with Hfq. We defined the exact coordinates of sRNAs, and confirmed that they are expressed at significant levels. We also determined the Hfq regulon in Salmonella, and reported the role of Hfq in controlling transcription of major pathogenicity islands, horizontally acquired regions, and the flagellar cascade. Hfq is reported to be a global regulator that affects the expression of almost a fifth of all Salmonella genes. Our new approach will allow sRNAs and mRNAs to be characterized from different genetic backgrounds, or from bacteria grown under particular environmental conditions. It will be valuable to scientists working on genetically tractable bacteria who are interested in the function of RNA-binding proteins and the identification of sRNAs. Loss of Hfq function also results in a non-motile phenotype for Salmonella and the deregulation of .70 abundant proteins, including the accumulation of outer membrane proteins (OMPs); the latter is accompanied by a chronic activation of the sE (s24)mediated envelope stress response [32,34]. Hfq has also been implicated in the control of Salmonella gene expression changes induced by the low gravity condition experienced during spaceflight [35]. Understanding how Hfq controls Salmonella gene expression at the pos (...truncated)