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Global Analysis of Small RNA and Mrna Targets of Hfq

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Global Analysis of Small RNA and Mrna Targets of Hfq Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 200350411111124Original ArticleA. Zhang et al.Global analysis of Hfq targets Molecular Microbiology (2003) 50(4), 1111–1124 doi:10.1046/j.1365-2958.2003.03734.x Global analysis of small RNA and mRNA targets of Hfq Aixia Zhang,1 Karen M. Wassarman,2 greatly expanded over the past few years (reviewed in Carsten Rosenow,3 Brian C. Tjaden,4† Gisela Storz1* Gottesman, 2002; Grosshans and Slack, 2002; Storz, and Susan Gottesman5* 2002; Wassarman, 2002; Massé et al., 2003). A subset of 1Cell Biology and Metabolism Branch, National Institute of these small RNAs act via short, interrupted basepairing Child Health and Development, Bethesda MD 20892, interactions with target mRNAs. How do these small USA. RNAs find and anneal to their targets? In Escherichia coli, 2Department of Bacteriology, University of Wisconsin, at least part of the answer lies in their association with Madison, WI 53706, USA. and dependence upon the RNA chaperone, Hfq. The 3Affymetrix, Santa Clara, CA 95051, USA. abundant Hfq protein was identified originally as a host 4Department of Computer Science, University of factor for RNA phage Qb replication (Franze de Fernan- Washington, Seattle, WA 98195, USA. dez et al., 1968), but later hfq mutants were found to 5Laboratory of Molecular Biology, National Cancer exhibit multiple phenotypes (Brown and Elliott, 1996; Muf- Institute, Bethesda, MD 20892, USA. fler et al., 1996). These defects are, at least in part, a reflection of the fact that Hfq is required for the function of several small RNAs including DsrA, RprA, Spot42, Summary OxyS and RyhB (Zhang et al., 1998; Sledjeski et al., Hfq, a bacterial member of the Sm family of RNA- 2001; Massé and Gottesman, 2002; Møller et al., 2002). binding proteins, is required for the action of many All of these small RNAs are believed to act by comple- small regulatory RNAs that act by basepairing with mentary pairing with target messages and Hfq has been target mRNAs. Hfq binds this family of small RNAs shown to promote annealing of OxyS and Spot42 RNAs efficiently. We have used co-immunoprecipitation with to their target mRNAs in vitro (Møller et al., 2002; Zhang Hfq and direct detection of the bound RNAs on et al., 2002). In addition to its role in facilitating small RNA genomic microarrays to identify members of this function, Hfq also has been found to contribute to regula- small RNA family. This approach was extremely sen- tion of ompA mRNA stability and the polyA tailing of some sitive; even Hfq-binding small RNAs expressed at low mRNAs (Hajndsorf and Regnier, 2000; Vytvytska et al., levels were readily detected. At least 15 of 46 known 2000). small RNAs in E. coli interact with Hfq. In addition, Hfq is a highly conserved protein encoded within many high signals in other intergenic regions suggested up bacterial genomes (Sun et al., 2002). It oligomerizes into to 20 previously unidentified small RNAs bind Hfq; a hexameric ring structure, and both sequence and struc- five were confirmed by Northern analysis. Strong sig- tural analyses show a significant similarity to archaeal and nals within genes and operons also were detected, eukaryotic Sm and Sm-like proteins integral to premRNA some of which correspond to known Hfq targets. splicing and RNA degradation complexes (Schumacher Within the argX-hisR-leuT-proM operon, Hfq appears et al., 2002). Hfq does not have a precise target sequence to compete with RNase E and modulate RNA process- but appears to bind unstructured AU rich sequences, fre- ing and degradation. Thus Hfq immunoprecipitation quently close to more structured RNA regions (Vytvytska followed by microarray analysis is a highly effective et al., 2000; Zhang et al., 2002; Brescia et al., 2003), sim- method for detecting a major class of small RNAs as ilar to, although not as specific as, the binding sites well as identifying new Hfq functions. defined for eukaryotic Sm and Sm-like proteins. We recently carried out a genome-wide search for new small RNAs in E. coli and identified 17 novel RNAs (Was- Introduction sarman et al., 2001). As a component of the character- The recognition of the roles of small, non-coding RNA ization of these new small RNAs, we tested for potential regulators in bacteria, archaea and eukaryotes has interactions with Hfq using a co-immunoprecipitation analysis. We found significant enrichment for eight of the Accepted 21 July, 2003. *For correspondence. E-mail novel small RNAs in the samples selected with Hfq- [email protected]; Tel. (+1) 301 402 0968; Fax (+1) 301 496 3875; specific antiserum, indicating they interact with Hfq or E-mail [email protected]; Tel. (+1) 301 496 3524; Fax (+1) 301 496 3875. †Present address. Computer Science Department, (Wassarman et al., 2001). Other small RNAs were either Wellesley College, Wellesley, MA 02481, USA. poorly enriched, or not detected among the selected © 2003 Blackwell Publishing Ltd 1112 A. Zhang et al. RNAs, indicating they bind Hfq inefficiently or not at all. Microarray detection of known small RNAs which bind Hfq To directly detect and identify more members of the Hfq- dependent family of small RNAs, we carried out microar- A total of 46 small RNAs have been described in E. coli, ray analysis of E. coli RNAs which co-immunoprecipitate as a result of earlier work as well as recent genome-wide with Hfq. To evaluate the reliability and sensitivity of this searches (Argaman et al., 2001; Rivas et al., 2001; approach, we first analysed 46 known small RNAs and Wassarman et al., 2001; Chen et al., 2002) (Table 1). We found that at least 15 specifically interact with Hfq, in began our analysis by evaluating the ability of these small agreement with and extending previous studies. Microar- RNAs to be detected in the Hfq co-immunoprecipitated ray results predicted the presence of 20 novel Hfq-bind- samples using the microarray method and rating as ing small RNAs; five were confirmed to be bound by Hfq described above. Seventeen RNAs were rated 5, four by Northern analysis. We also found that Hfq associates RNAs were rated 4, and the rest had lower scores. Sev- with a number of mRNAs and operon mRNAs, suggest- eral of these 46 small RNAs had previously been tested ing this approach also may be useful for identification of for their ability to bind Hfq; 12 were shown to efficiently RNAs that are targets of Hfq, either directly or indirectly interact with Hfq, five did not bind Hfq and one gave partial through the action of an associated small RNA. Finally, binding (Sledjeski et al., 2001; Wassarman et al., 2001; we have identified an association of Hfq with precursors Møller et al., 2002; Zhang et al., 2002). These data pro- of the proM tRNA, suggesting yet other roles for Hfq vided a reference set for evaluating the sensitivity and within the cell. specificity of the microarray approach. Of the 12 previ- ously identified Hfq-binding RNAs, 11 were rated 5 in our microarray tests. Of those previously found not to bind Hfq Results or only show partial binding, all except tmRNA (see below) had a score of 4 or lower. Microarray detection of RNAs which To directly assess Hfq binding to seven previously co-immunoprecipitate with Hfq untested small RNAs, Northern analysis was carried out Wild-type cells were grown under three different condi- on the RNA extracted from Hfq immunoprecipitation sam- tions, LB (exponential phase and stationary phase) and ples (Fig. 1). Three of the four RNAs which were rated 5 minimal glucose medium. Extracts from these cells were or 4 were confirmed to bind Hfq (DicF, SraD and MicF) prepared and subjected to immunoprecipitation with either and none of the three RNAs tested that were rated 3 or Hfq-specific serum or control preimmune serum. Immuno- lower (RydB, IS092, and RNase P) were positive for Hfq precipitated RNAs were identified by direct hybridization binding. Thus, for previously detected small RNAs, a rat- on DNA microarrays; these microarrays carry 15 oligonu- ing of 5 was a good predictor of Hfq-binding. cleotide probes (25-mers) within each gene and most There are two exceptions where we see microarray intergenic (Ig) regions. Hybridization of RNAs to oligonu- scores of 5, yet do not find specific interactions with Hfq cleotide probes was detected directly with antibodies spe- as assessed by Northern analysis, 4.5S (Fig. 1) and cific for RNA:DNA hybrids (see Experimental procedures). tmRNA (Wassarman et al., 2001). Notably, these two This novel method significantly improved the sensitivity of RNAs are present at very high levels within the cell, and detection and avoided problems inherent in the labelling therefore it is possible these RNAs give high scores on of small, structured RNAs. Two separate experiments microarrays as a result of their abundance. High signal were carried out in LB (coded E1, E2 for the exponential was observed for both RNAs in the immune and preim- phase samples and S1, S2 for the stationary phase sam- mune samples (Experimental procedures and data not ples); the experiment in minimal glucose media (M) was shown), and 4.5S is detected in both the anti-Hfq and a single trial. preimmune samples upon overexposure of the Northern Corrected signals for individual probes were calculated, blot. Alternatively, it is possible that the 4.5S and tmRNA and the regions with two or more adjacent probes above or their precursors do exhibit transient or low-efficiency a given expression level were rated as described in Exper- binding to Hfq. We note that the RNase P and 6S RNAs, imental procedures. If the average signal for a group of which also are abundant, did not give a high score in the probes was equal to or greater than 10 000 in two dupli- microarray, even before correction for the preimmune cate experiments (E1 and E2 or S1 and S2) or in the samples (data not shown), although they too can be single minimal media experiment (M), the region was detected in overexposed Northern blots in both immune rated 5.
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