RNA Biology

ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: http://www.tandfonline.com/loi/krnb20

Experimental tools to identify RNA-protein interactions in Helicobacter pylori Renate Rieder, Richard Reinhardt, Cynthia Sharma & Jörg Vogel To cite this article: Renate Rieder, Richard Reinhardt, Cynthia Sharma & Jörg Vogel (2012) Experimental tools to identify RNA-protein interactions in Helicobacter pylori, RNA Biology, 9:4, 520-531, DOI: 10.4161/rna.20331 To link to this article: http://dx.doi.org/10.4161/rna.20331

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Research paper

RNA Biology 9:4, 520-531; April 2012; © 2012 Landes Bioscience

Experimental tools to identify RNA-protein interactions in Helicobacter pylori Renate Rieder,1 Richard Reinhardt,2 Cynthia M. Sharma3,* and Jörg Vogel1,* Institute for Molecular Infection Biology; University of Würzburg; Würzburg, Germany; 2Max Planck Genome Centre Cologne; Cologne, Germany; 3 Research Centre for Infectious Diseases (ZINF); University of Würzburg; Würzburg, Germany

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Key words: Helicobacter pylori, small RNA, Hfq, post-transcriptional control, RNA binding proteins, co-immunoprecipitation, affinity chromatography, RNA-seq

Helicobacter pylori, one of the most prevalent human pathogens, used to be thought to lack small regulatory RNAs (sRNAs) which are otherwise considered abundant in all bacteria. However, our recent analysis of the primary transcriptome of H. pylori discovered an unexpectedly large number of sRNAs, and suggested that this model organism also uses riboregulation to control the expression of its genes. Nonetheless, whereas most enterobacterial sRNAs require the RNA chaperone Hfq for function, Epsilonproteobacteria including H. pylori seem to have no Hfq homolog, which prompted us to search for other auxiliary proteins in sRNA-mediated regulation. Therefore, we have developed two orthogonal methods to isolate and investigate in vivo and in vitro assembled RNA-protein complexes in H. pylori: (1) an affinity chromatography strategy based on aptamer-tagged sRNAs of interest to identify their protein binding partners; and (2) a rapid method for chromosomal FLAG-tagging of proteins to facilitate co-immunoprecipitation of associated RNA species. Using these methods, we have identified RNA-protein interactions between the ribosomal protein S1 and various mRNAs and sRNAs of H. pylori. Moreover, both methods reported a stable RNA-protein complex between the abundant HPnc6910 sRNA and HP1334, a protein of unknown function that is encoded downstream of HPnc6910. Given that 50% of all bacteria may lack Hfq, our methods can be useful to identify RNA-protein interactions in a wider range of bacterial pathogens.

© 2012 Landes Bioscience. Do not distribute. Introduction

Helicobacter pylori is a microaerophilic, Gram-negative-e proteobacterium which is under intense investigation due to its contributions to gastritis, peptic ulcer disease and gastric malignancy. Although its genome sequence has been known for long,1 our understanding of gene regulation in this major human pathogen has been hampered by a lack of a satisfactory repertory of molecular analysis tools. Compared with other Gram-negative model bacteria such as Escherichia coli or Salmonella, gene expression control in Helicobacter seems to rely on a limited number of mechanisms. Several important alternative sigma factors are missing, and there are only very few transcription factors and two-component systems.1,2 Furthermore, almost nothing is known regarding post-transcriptional regulation in H. pylori, and except for the ubiquitous housekeeping RNAs (4.5S RNA, RNase P, and tmRNA), none of the many small RNA species known in enterobacteria are conserved in H. pylori. In addition, the common RNA chaperone Hfq which facilitates the base pairing of many regulatory RNA molecules to target mRNAs in many other bacteria seems absent from the entire ε-subdivision.3,4 Given the above observations, H. pylori was increasingly perceived as an organism that lacks riboregulation.5 However, our

recent deep-sequencing based analysis of the primary transcriptome of H. pylori strain 26695 dramatically challenged this perception; using a novel differential RNA-sequencing (dRNA-seq) approach, we discovered > 60 sRNAs and a large number of cisencoded antisense transcripts in Helicobacter.6 The functions and molecular mechanisms of these new sRNAs remain to be elucidated. By way of inference from other Gram-negative bacteria, one may speculate that many of these sRNAs act on mRNAs, and depend on a common RNA-binding protein with a function analogous to that of Hfq. The identification of a protein with general Hfq-like function could facilitate the same global approaches involving co-immunoprecipitation (co-IP) and analysis of the associated RNA by microarray or deep sequencing7-9 that have tremendously advanced our understanding of sRNA functions in Salmonella and E. coli.10 Furthermore, several other bacterial proteins that interact with sRNAs have been described (reviewed in ref. 11). For example, the almost ubiquitous RNA-binding protein, CsrA (carbon storage regulator), acts as a post-transcriptional regulator of mRNAs and its activity is inhibited by CsrB-like sRNAs in a wide range of bacteria.12,13 A homolog of this protein has been identified and investigated in H. pylori;14 however, neither CsrBlike sRNAs nor the regulatory mechanisms of CsrA are known in Helicobacter. As another example, protein HP0958 was recently

*Correspondence to: Cynthia M. Sharma and Jörg Vogel; Email: [email protected] and [email protected] Submitted: 03/08/12; Revised: 04/09/12; Accepted: 04/10/12 http://dx.doi.org/10.4161/rna.20331 520

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Research paper

shown to be essential for flagellum biogenesis and to act as a translational regulator of flagellin mRNA.15 Nonetheless, the only RNA-protein complex which has be studied in detail in H. pylori consists of the essential genes tmRNA and the co-factor smpB, which cooperate to rescue stalled ribosomes and facilitate stress responses in this organism.16,17 Thus, it is desirable to have experimental means to identify new RNA-protein interactions in H. pylori in a generic fashion. Several strategies for the investigation of RNA-protein interactions have been developed over the years (reviewed in ref. 18). Of these, we have here adapted the in vitro approach by Winbichler et al.,19 which combines aptamer-tagging of a-sRNA of interest with affinity chromatography and subsequent mass spectrometry of the retained sRNA-binding proteins. In lieu of the streptomycin aptamer tag in the original study in E. coli, however, we chose the MS2 aptamer which had been used successfully to isolate RNA-protein complexes from Salmonella lysates.20 As an orthogonal strategy we used co-IP combined with deep sequencing of RNA (RNA-seq) to validate identified RNAprotein interactions in vivo and to assess the complement of transcripts bound by a protein of interest on a global scale. The combination of co-IP with RNA-seq previously helped to discover many new sRNA and mRNA binding partners of Hfq in Salmonella.8,9 In order to be able to perform co-IP with RNAbinding proteins in H. pylori, we first developed a rapid protocol for chromosomal tagging of proteins (reading frames) with a 3xFLAG epitope. Based on these methods we have identified novel examples of RNA-protein complexes in H. pylori. These include interactions between the ribosomal protein S1 (a.k.a. HP0399) with several mRNAs and sRNAs as well as a stable complex between a protein of unknown function, HP1334, and the abundant sRNA HPnc6910, which is derived from the 5′ UTR of HP1334. These experimental tools promise to be generally useful for the isolation and identification of RNA-protein complexes of H. pylori and related pathogens.

Following several wash steps to remove unspecifically bound cellular components, the tagged RNA and its associated proteins are eluted under native conditions with a maltose-containing buffer that disrupts the MBP-amylose interaction. Finally, the co-purified proteins are analyzed by mass spectrometry. Aptamer tagging and affinity chromatography of abundant sRNAs. We selected several abundant sRNAs, namely HPnc5490, HPnc6670, HPnc2420, HPnc7830, HPnc0580, HPnc1980, and HPnc6870, for MS2 tagging; all of these sRNAs had been easily detectable on northern blots with total RNA of H. pylori strain 26695.6 The DNA templates for T7 transcription of the aptamer-tagged sRNAs were generated by overlap PCR (see Material and Methods). We generally tagged the sRNA of interest at the 3′ end, after the intrinsic terminator, i.e., the part we assume not to be involved in RNA-protein interaction. Tagging after the terminator did not lead to any premature transcription termination since the T7 RNA polymerase does not recognize the weak terminators of H. pylori. In addition, for some sRNAs we also generated a 5′ tagged version to minimize the risk of missing proteins that would bind to the 3′ end of the sRNA. For example, we have constructed 5′ and 3′ tagged versions of the ~140-nt long HPnc6910 sRNA (Fig. 2A). The resulting RNA sequences of 5′ or 3′ tagged versions of this sRNA are shown in Figure 2B. Affinity chromatography followed by northern blot analysis showed that either tagged variant of HPnc6910 can be stably recovered from the lysate (Fig. 2C). Quantification of the signals in the lysate (input) and the eluted fractions show that 10 to 30% of the input RNA are recovered. Comparative affinity purification with the MS2 tag as control RNA revealed selective enrichment of two ~25 kDa and ~58 kDa proteins which were only recovered with the tagged HPnc6910 sRNAs (Fig. 2D). Mass spectrometric analysis of protein bands excised from the gel suggested these proteins to be the 26 kD protein HP1334 of unknown function, and the 63 kDa ribosomal protein S1. In subsequent analysis of several other abundant sRNAs, we did not observe a co-purification of HP1334, indicating a specific RNAprotein interaction between HPnc6910 and HP1334. However, most of the other sRNAs also brought down the S1 protein, suggesting that S1 constitutes a rather general sRNA binding protein (Fig. S1). In vivo validation of RNA-protein interactions. The use of in vitro transcribed, tagged RNAs in lysates, rather than in vivo expression of the tagged sRNA of interest, bears the risk of isolating non-natural complexes. Moreover, the in vitro transcribed RNAs are typically used in excess over endogenous levels of sRNAs which might result in unspecific binding of proteins. Therefore, to validate the above identified candidate interactions, we used co-IP of the proteins in questions and inspected the bound RNA species by northern blot analysis and deep sequencing (Fig. 1B). With respect to affinity purification of tagged RNAs, this strategy represents the reverse approach wherein the RNA-binding protein rather than the RNA is tagged. The co-IP followed our previously described approach for the isolation of sRNA and mRNA binding partners of 3xFLAG-tagged Hfq in Salmonella.8,9 First, H. pylori cells expressing the protein of

© 2012 Landes Bioscience. Do not distribute. Results Affinity purification strategy to identify proteins interacting with H. pylori sRNAs. To identify sRNA-associated proteins in H. pylori, we have established an in vitro affinity chromatography approach that utilizes a MS2 aptamer tag that is added to a sRNA of interest (Fig. 1A). The MS2-aptamer very specifically binds to a fusion protein consisting of the coat protein of phage MS2 and the maltose binding protein (MBP), the latter of which permits immobilization on a column.21 Beginning with in vitro synthesis of the aptamer-tagged sRNA using T7 RNA polymerase, the affinity chromatography is performed as follows (Fig. 1A): The tagged sRNA is incubated with a whole-cell lysate of H. pylori to allow for binding of interacting cellular proteins. Next, the lysate is applied to the column in which the bait protein MS2-MBP is non-covalently coupled to amylose beads via the MBP moiety of the fusion protein. The RNA-protein complexes are retained by interaction of the aptamer with the MS2 coat protein part.

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© 2012 Landes Bioscience. Do not distribute. Figure 1. Strategies for the isolation of RNA-protein complexes in H. pylori. (A) Aptamer tagging of sRNAs combined with affinity chromatography is used for the identification of protein interaction-partners of specific sRNAs. The MS2 tag of the in vitro transcribed RNAs is shown in red. (B) Coimmunoprecipitation of epitope-tagged RNA-binding proteins combined with deep sequencing provides a global picture of RNAs that are associated with the tagged proteins. The 3xFLAG tag is indicated as a red circle.

interest with a 3xFLAG tag from the chromosome, are collected and lysed. Next, α-FLAG antibody and then Protein A sepharose beads are sequentially added to the lysate to specifically immobilize the desired RNA-protein complexes. After washing the beads, the complexes can be recovered under denaturing conditions by phenol-extraction. Proteins residing in the organic phase can be analyzed by western blotting and Coomassie staining. The co-immunoprecipitated RNA, found in the aqueous phase, is converted into cDNA libraries for analysis by deep sequencing. In addition, RNA samples of the different steps of the co-IP are investigated on northern blots. Chromosomal epitope-tagging of proteins in H. pylori. As a prerequisite of co-IP, proteins need to be endowed with an

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epitope tag, ideally by addition to the chromosomal open reading frame. Unlike in many other bacteria in which chromosomal epitope tagging can be easily achieved using the lambda red recombination system,22 no simple and fast method for FLAG-tagging of proteins in the H. pylori chromosome was available. Moreover, the lambda red system has as yet not been shown to work in H. pylori. Therefore we designed a new method based on a two-step overlap PCR to generate a DNA fragment, which can be directly transformed into naturally competent H. pylori, and introduces the 3xFLAG tag at the C-terminus via homologous recombination into the chromosome (Fig. 3A). Following this strategy, we tagged the two above identified candidate proteins, HP1334 and ribosomal protein S1. Western blot analysis, as part of the

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© 2012 Landes Bioscience. Do not distribute. Figure 2. Affinity chromatography of MS2-tagged HPnc6910 sRNA. (A) Genomic location and predicted secondary structure of the abundant sRNA HPnc6910. Expression of the ~140 nt-long HPnc6910 RNA is shown by northern blot analysis (black arrow). (B) Sequences and predicted secondary structures of in vitro transcribed 5′- and 3′-MS2 aptamer-tagged HPnc6910 variants. The sequence of the MS2 tag is indicated in red and spacer nucleotides neither belonging to the sRNA nor to the tag in gray, respectively. (C) RNA fractions of the different steps of affinity purification were analyzed by northern blot experiments. For the in vitro transcribed (ivt) RNA, 1 pmol was loaded. The amount of RNA in the lysate, flow through and wash sample correspond to 2 pmol and to 10 pmol in the eluate. (D) Protein fractions of the different steps of affinity purification were separated on an SDS-gel and stained with silver. Prominent bands close to 25 kDa and 58 kDA were excised and analyzed by mass spectrometry. Loaded protein samples correspond to 0.025 OD600 of bacterial culture for the lysate, flow through and wash fraction and to 3.5 OD600 for the eluate, respectively.

co-IP experiments showed, that both tagged proteins, HP1334– 3xFLAG and S1–3xFLAG, are stably expressed and detectable in the lysate (Fig. 3B, lanes 1 and 2). Moreover, 10 to 25% of the tagged protein can be isolated from the lysate by immunoprecipitation. Additional Coomassie staining of denaturing gels further confirmed the enrichment of the tagged proteins by the procedure (Fig. S2). Northern blot analysis of RNA samples isolated from co-IP experiments of H. pylori wild type, harbouring the untagged protein version, and HP1334–3xFLAG tagged strains, showed that only in the tagged strain the sRNA HPnc6910 is enriched in the co-IP sample (Fig. 3C, lane 5). In contrast, the housekeeping 6S RNA, serving as a control, was recovered (at

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low levels) in both co-IP experiments. Thus, consistent with the previous affinity chromatography, the co-IP experiment indicates a specific interaction between HPnc6910 sRNA and HP1334. Global analysis of additional RNAs isolated by co-IP using deep sequencing. For genome-wide identification of RNAs bound by proteins HP1334 and S1 in vivo, we subjected the RNA samples from the co-IP experiments to deep sequencing. To this end, RNA from either of the two tagged strains and from the untagged wild-type strains as negative control was converted to cDNA and sequenced using the 454 technology (Table S1). After mapping of the cDNA reads of the individual co-IP libraries to the genome sequence of strain H. pylori 26695,

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The enrichment patterns for ribosomal protein S1 were much different: dozens of RNAs were enriched with the FLAGtagged S1 protein (Fig. 4B; Fig. S3B); the most prominent peaks correspond to RNase P RNA and 6S RNA, but there were also several other transcripts including many mRNAs that were strongly enriched in the S1 co-IP library (Tables S2– S3). Surprisingly, none of the tagged sRNAs that pulled down S1 in the affinity chromatography (Fig. S1) shows significant enrichment in the in vivo co-IP experiments. For example, affinity chromatography experiments with the aforementioned HPnc6910 sRNAs had suggested interactions with both HP1334 and S1 (Fig. 2D); however, this sRNA was found to be enriched only in the co-IP with HP1334 and not with S1 (Fig. 5A). Thus, either HPnc6910 does not interact with the S1–3xFLAG protein in vivo, or the interaction is so labile that it only withstands the affinity chromatography but not the co-IP procedure. Functional interaction of protein HP1334 with sRNA HPnc6910. Having found evidence for an HPnc6910-HP1334 RNA-protein interaction both in vitro and in vivo, we were interested whether HP1334 influences the expression of HPnc6910 sRNA in vivo. Therefore, we used our new overlap PCR strategy to construct a strain that contains a start codon mutation (ATG→CTG) in the HP1334–3xFLAG strain (see Materials and Methods section). We analyzed protein and RNA levels during growth in liquid culture, comparing the mutant strains to both the FLAG-tagged strain and the parental WT strain (Fig. 5B). As expected nearly no HP1334–3xFLAG protein was expressed by the strain with the start codon mutation. Interestingly, this reduction in protein synthesis was accompanied by an almost total loss of a signal for the HPnc6910 sRNA, suggesting that HP1334 is needed for stable expression of the HPnc6910. Since HPnc6910 likely constitutes part of the 5′ UTR of the HP1334 mRNA, this suggests that HP1334 might auto-regulate its own expression by acting on the 5′ region of its own transcript.

© 2012 Landes Bioscience. Figure 3. Chromosomal epitope-tagging of HP1334 and ribosomal protein S1 and subsequent co-IP experiments. (A) A cassette was designed and generated by overlap PCR to add a 3xFLAG tag to genes on the chromosome via homologous recombination. The desired DNA fragment is produced by overlap PCR. (B) Protein fractions of the different steps of the co-IP of HP1334–3xFLAG and S1–3xFLAG analyzed by western blot. Loaded protein samples correspond to 0.025 OD600 of bacterial culture for the culture, lysate, supernatant (SN) and wash fraction; the co-IP corresponds to 0.2 OD600. (C) RNA fractions of the different steps of the co-IP of HP1334–3xFLAG were analyzed by northern blot. As a control for unspecific binding, a co-IP experiment with wild-type H. pylori 26995 lysate (WT) was performed and analyzed in parallel. Loaded RNA samples correspond to 2 OD600 of bacterial culture for the culture, lysate, supernatant (SN) and wash fraction; the co-IP corresponds to 20 OD600.

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we searched for enrichment of transcripts in the co-IP samples of the FLAG-tagged strains as compared to the untagged wild type, to exclude sequences that represented unspecific binding (Fig. 4). The data obtained with the HP1334–3xFLAG protein (Fig. 4A; Fig. S3A) showed the most prominent peak (highest enrichment) at approximate position 1,395,000 in the genome, which corresponds to sRNA HPnc6910 (for quantification see Tables S2–S3). This confirms that the interaction of HP1334 with HPnc6910, which was initially suggested in vitro by affinity chromatography with the sRNA, is also likely to occur in vivo. The association of HP1334 with the upstream transcribed HPnc6910 RNA seems to be very specific (Fig. 4A) because no other RNAs were strongly enriched with the FLAG-tagged HP1334 protein (Fig. 3A; Tables S2-S3). Detailed comparison of the sequencing reads for the co-IP experiments of HP1334– 3xFLAG with the whole transcriptome data6 shows that the 5′ region of HPnc6910 was most highly enriched, indicating that this might be the region that becomes protected during binding of protein HP1334 (Fig. 5A).

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Discussion Several methods have been described for the isolation of RNP-complexes in enterobacteria, such as E. coli and Salmonella.7,19,20,23,24 Due to a lack of molecular biology tools, e.g., stable plasmids or inducible promoters, some of these methods cannot be easily adapted to other bacteria. In this method paper, we present two strategies for the isolation and characterization of RNA-protein complexes in the pathogenic e-proteobacterium Helicobacter pylori. First, in vitro transcribed MS2-aptamer-tagged sRNA variants were incubated with whole cell lysates of H. pylori and subjected to affinity chromatography. The use of in vitro transcribed RNAs has the advantage that no genetic manipulation or expression from a plasmid is required. Therefore, this strategy is also suitable for organisms, which are not genetically tractable. Additionally, it is easily feasible to screen in parallel several in vitro transcribed sRNA candidates for protein binding partners. Besides the above described MS2-aptamer tag, we also tested 5′-biotinylated sRNAs

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Figure 4. RNA-seq analysis of RNAs co-immunoprecipitated with 3xFLAG tagged variants of HP1334 and ribosomal protein S1. (A) cDNA reads mapped to the genome corresponding to co-purified RNA from the co-IP experiment of HP1334–3xFLAG (blue). As a control for unspecific binding, a co-IP with wild-type H. pylori 26995 lysate was performed and analyzed in parallel (black). (B) The same as in (A) but the co-IP experiment of S1–3xFLAG (red) was compared with the control (black). The RNAs which show the highest enrichment in the co-IPs with the tagged proteins are indicated at their genomic location. RNAs which are specifically enriched in the 3x-FLAG coIPs compared to the control coIP are labeled in red and those which show similar recovery in boths co-IPs in black, respectively. The y-axis represents a relative score for the number of mapped cDNAs and is scaled according to the RNA enrichment factor (for details see Materials and Methods).

for affinity chromatography but those RNAs appear to be rather unstable in a lysate of H. pylori (data not shown). By contrast, the MS2 aptamer allowed us to recover the tagged RNA in high yields (between 10 to 30%). Moreover, we were able to recover

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associated RNA-binding proteins along with the aptamer tagged RNAs using this strategy. For example, a protein of unknown function, HP1334, specifically co-purified with HPnc6910, a sRNA which is derived

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Figure 5. HPnc6910 associates with HP1334 in vivo but not with ribosomal protein S1. (A) cDNA reads that were mapped to the genome in the region of HPnc6910 and HP1334. Reads corresponding to co-IP experiments with HP1334–3xFLAG (blue) and S1–3xFLAG (red) are compared with the control co-IP with the untagged wild-type strain for unspecific binding (black). Moreover, reads corresponding to whole transcriptomes libraries (gray, with (+) or without (-) terminator exonuclease treatment TEX) from RNA harvested during mid-log growth are shown. 6 The TEX-treated cDNA libraries correspond to primary transcripts, whereas the untreated cDNA library reflects primary and processed transcripts. (B) HPnc6910 and HP 1334 expression in the HP1334–3xFLAG strain was compared with the expression in the corresponding strain with an ATG to CTG mutation and in the wildtype H. pylori 26695 by northern blot and western blot analysis.

from the 5′ UTR of the HP1334 mRNA (Fig. 2); ribosomal protein S1 co-purified with several tagged sRNAs (Fig. S1). Also in E. coli, ribosomal protein S1 was shown to co-purify in vitro with several sRNAs that were tagged with a streptomycinbinding RNA aptamer.19 However, since S1 has been shown to interact with Hfq,25 the co-purification of protein S1 along with the aptamer-tagged sRNAs might be indirectly mediated via a protein-protein interaction in E. coli. For the experiments

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presented here, however, we do not assume indirect binding of the ribosomal protein S1 to the tested sRNAs, at least not via a protein, because we could only see S1—and no other proteins of H. pylori—to be enriched by affinity chromatography (Fig. S1). Nonetheless, it is important to test whether the sRNA interactions with proteins HP1334 or S1, as suggested by the in vitro approach, also form in vivo. Therefore, we have established a second method based on co-IP of chromosomally tagged

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RNA-binding proteins combined with RNA-seq.8,9 To this end, we have developed a rapid method involving a two-step overlap PCR to generate 3xFLAG tagged genes in the chromosome of H. pylori. Note that this chromosomal FLAG-tagging approach will be useful beyond the presented co-IP experiments, since it will facilitate a variety of other applications in H. pylori, e.g., expression analysis of proteins based on western blots or detection of shorter and longer protein variants. Compared with the TAP-tag, which has previously been used for tandem affinity purification, e.g. of the urease complex in H. pylori,26 the 3xFLAG tag epitope has the advantage that it is very small and, thus, less likely to disturb protein function or localization, and to interfere with the formation of RNA-protein complexes. Based on our two approaches, we have identified in vitro and in-vivo a specific RNA-protein complex between the abundant sRNA HP6910 and a protein of unknown function, HP1334. Unfortunately, the function of HP1334 in H. pylori is not known yet. Since the HP1334 gene is conserved in only a few H. pylori strains such as 26695 or G27, and disrupted or absent in many others (Fig. S4A–B), it is unlikely that HP1334 represents a general RNA binding protein. It rather seems that the protein was horizontally acquired together with the upstream sRNA, which is also reflected by the presence of predicted homologs of HP1334 in diverse bacteria, e.g., Campylobacter lari, Aeromonas salmonicida, Neisseria menigitidis (Fig. S4C). HP1334 shows homology to the LabA-like superfamily, which is composed of a well-conserved group of bacterial proteins with as yet undefined function. LabA, a member from Synechococcus elongatus PCC 7942, has been shown to play a role in cyanobacterial circadian timing.27 However, our preliminary results of gene disruption of HP1334 in H. pylori does not support a major influence on protein expression in H. pylori, at least under standard growth conditions (data not shown). The next step will be to investigate potential functions of the protein, which in turn may give away the function of the HP6910 sRNA, which appears to be one of the most abundant small non-coding RNAs in H. pylori.6 Whereas the in vivo experiments confirmed a specific RNAprotein interaction between HPnc6910 and HP1334, the interaction of ribosomal protein S1 with this and other sRNAs suggested by affinity chromatography failed to be confirmed by the reciprocal co-IP experiment (Tables S2–S3). This discrepancy of in vivo and in vitro assembled complexes might be due to the excess of in vitro transcribed aptamer tagged sRNAs used in affinity chromatography or different stabilities of the complexes in the two different approaches. The use of sRNAs in large excess bears the risk of causing false-positive results because it may force S1 into complexes, which would not be formed with natural concentrations of the diverse binding partners. In general, the stability of the complexes and specificity of co-purifying interaction partners could be improved by introducing a crosslinking step prior to the isolation of RNP-complexes (reviewed in ref. 18). Although the interaction between S1 and the sRNAs that were used for aptamer tagging was not visible in the S1 co-IP, the RNA-seq analysis of the RNAs that were co-immunoprecipitated with S1–3xFLAG revealed dozens of other RNAs that are associated with S1 in H. pylori in vivo (Tables S2–S3). Most of

the enriched RNAs belong to mRNAs which are probably bound by S1 during translation. A first proteomics analysis of whole cell protein expression patterns on 1D SDS-PAGE shows that several proteins, e.g., UreA, SodB, and TrxA, are differentially expressed in a ΔS1 deletion mutant strain, as compared to the wild-type strain (Fig. S5). Moreover, the mRNAs of some of the differentially expressed proteins, e.g., ureA, sodB and trxA, were also enriched in the S1 co-IP library, indicating that they are directly bound by S1 in vivo (Fig. S3 and Table S3). Thus, the co-IP approach identified promising candidates for future functional characterizations of S1 targets in this pathogen. Until now, no detailed studies on the diverse functions of ribosomal protein S1 itself in H. pylori have been performed. Comparable to Hfq, S1 plays pleiotropic roles in a wide range of RNA transactions in diverse bacteria (reviewed in ref. 28). For example, this ribosomal protein is needed for efficient translation of most mRNAs27,28 and has been suggested to be implicated in transcription and mRNA stabilization.29-32 Using Far-Western assays, Feng et al. have shown that the S1 protein interacts with RNase E and PNPase, which are part of the degradosome in E. coli.33 Interestingly, H. pylori lacks a homolog of this major endoribonucleases E1; however, in a large-scale protein-protein interaction map of H. pylori a potential interaction between S1 and RNase J (HP1430) was detected.34 This ribonuclease has a key role in RNA maturation and turnover in Gram-positive bacteria such as Bacillus subtilis.35 Furthermore, S1 from E. coli has also been shown to be important for the endoribonucleic function of T4-encoded RegB and for replication of phage RNA mediated via QB replicase.36,37 Besides this, the S1 protein has a controversially discussed role in trans-translation which is mediated via TmRNA (reviewed in ref. 28). Whether Hfq and S1, which both preferentially bind to A/U-rich sequences but show a different chaperone activity in vitro,38 are just working independently or, in some cases, are also performing overlapping functions in the cell is still unclear.28 Although S1 homologs of H. pylori and E. coli share only ∼30% overall sequence identity, both of them are composed of six homologous S1 domains, D1-D6 (Fig. S6). Most of the amino acids of the consensus sequences within the five β-strands, which build up the b-barrel of the S1-motif, are conserved in H. pylori. 39 However, the sequence identity varies between the six domains: 15% for D1, 26% for D2, 34% for D3, 36% for D4, 39% for D5 and 18% for D6, respectively. The domains D1 and D2, which are involved in ribosome binding, are less conserved compared to the domains D3-D5, which are involved in the interactions with mRNAs in E. coli, indicating that the functions of the individual S1 domains in H. pylori and E. coli could be slightly different.40 Furthermore, the sixth domain, which is also thought to be involved in RNA binding but which is dispensable for translation initiation in E. coli, is also less conserved in H. pylori. Another remarkable difference between S1 from H. pylori and E. coli is the length of their 5′ leader sequences. Expression of S1 is autoregulated in E. coli by binding of S1 to its leader region.41 At least 90 nucleotides upstream of the start codon are required for this autocontrol and the secondary structure of the leader region is highly conserved in most

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γ-proteobacteria. In H. pylori, the transcriptional start site was mapped ~36 nt upstream of the start codon of S1,6 suggesting a different regulatory mechanism for rpsA expression in this ε-proteobacterium. Moreover, a transposon mutagenesis study indicated that rpsA is a non-essential gene in H. pylori42 and in line with this we could easily delete S1 in H. pylori (Fig. S5). S1 is also not essential in B. subtilis and has been shown to be absent from ribosomes of Gram-positive bacteria with a low G/C content.39,40,43 In contrast, S1 cannot be deleted in E. coli since it is essential for translation of most genes.27,44 How far these differences are due to different functions or target genes of S1 in H. pylori compared with the model organism E. coli is still an open question. Furthermore, it will be interesting to see, whether S1 in H. pylori—an Hfq lacking organism—could take over functions which are attributed to Hfq in E. coli. Since S1 has been shown to act as an RNA chaperone and resolve RNA structures in a non-sequence specific way,38 it has the potential to act as an auxiliary factor in riboregulation in H. pylori. Especially since H. pylori has an A/U-rich genome (38.9% GC-content compared to 50.8% of E. coli), S1 could play a role as a general RNA binding protein by binding to A/U-rich parts in many transcripts without strong specific interactions. Taken together, using two new methods for the isolation and characterization of RNA-protein complexes in H. pylori we have identified novel protein-RNA complexes in this organism. The herein developed molecular biological tools will be useful for the investigation of RNA-based regulation in Helicobacter and also other bacterial pathogens.

JVO-5095/5096 for the PCR on genomic DNA; for S1 use JVO5148/5149) with part of the aphA-3′ cassette. In the first overlap PCR step, products A and B were joined to AB and C and D to CD, respectively. For AB, corresponding PCR products were combined in equimolar amounts and PCR reactions using oligos JVO-5140/5144 for HP1334 and JVO5146/5144 for S1 were performed. For CD, JVO-5068/5096 for HP1334 and JVO-5068/5149 for S1 were used. In the second overlap PCR step, products AB and CD were joined to ABCD by PCR amplification using JVO-5140/5096 for HP1334 and JVO5146/5149 for S1, respectively. For the mutation of the ATG start codon to CTG in the HP1334–3xFLAG strain (JVS-7107), a DNA fragment was generated by overlap PCR of two PCR products, E and F; (E) contains a 350 bp region upstream of the start codon, the mutation at the start codon and a part downstream (PCR with JVO5212/7272 on genomic DNA of strain JVS-7033); (F) starts with the mutated C and contains HP1334–3xFLAG together with aphA-3′ kanamycin resistance cassette as described above and a 500 bp region downstream of the STOP codon of HP1334 (PCR with JVO-5947/5096 on genomic DNA of JVS-7033). In the overlap PCR step, products E and F were joined to EF by mixing the corresponding PCR products in equimolar amounts and performing PCR using oligos JVO-5212/5096. For the sequential deletion of ribosomal protein S1, three PCR products G, C and H were joined by overlap PCR; product G varies for the mutant strains S11–5, S11–2 and ΔS1, products C and H are the same for all three mutants. In detail the three PCR products were gained as follows: (G) 500 bp region of the S1; contains a part of the aphA-3′ cassette and was made by PCR with JVO-5369/5370 on genomic DNA of WT for strain S11–5, with JVO-5367/5368 for strain S11–2 and with JVO-5365/5366 for strain ΔS1; (C) the aphA-3′ kanamycin resistance cassette was amplified with primers JVO-5068/HPKterm on genomic DNA of the HPnc5490 deletion strain (see above); (H) a 500 bp region in the 3'¢part of S1 (~250 nt upstream and 250 nt downstream of the STOP codon); contains a part of the aphA-3′ cassette; JVO5363/5364 were used for PCR on genomic DNA of WT. DNA fragments (ABCD, EF or GCH) were incorporated into wild-type Helicobacter pylori 26695 genome via natural transformation according to.46 All mutations were verified by sequencing. Bacterial growth condition. H. pylori was grown on GC agar plates containing 10 µg/ml vancomycin, 5 µg/ml trimethoprim and 1 µg/ml nystatin at 37°C under microaerophilic conditions. Kanamycin (8 µg/ml) was added for selection when needed. For growth in liquid medium, BHI (brain-heart infusion medium) containing 10% fetal calf serum (FCS), 10 µg/ml vancomycin, 5 µg/ml trimethoprim and 1 µg/ml nystatin was used. Flasks were incubated at 37°C under microaerophilic conditions and agitated (140 rpm) until the culture reached the desired OD600. In-vitro transcription of aptamer tagged RNA HPnc6910. The DNA templates were generated via overlap PCR of two PCR products sharing the same sequence elements at the corresponding ends. DNA template preparation is described in detail for HPnc6910, but works accordingly for the other sRNAs

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Materials and Methods

Oligodesoxyribonucleotides. DNA oligonucleotides used in this study are listed in Table S4. Bacterial strains. Helicobacter pylori strains used in this study are listed in Table S5. H. pylori strain 26695 was used throughout this study. Chromosomal mutations were generated by natural transformation of a DNA fragment and subsequent homologs recombination. A graphical representation of all mutant strains is shown in Figure S7 which also illustrates the primer binding sites for the generation of the DNA fragments. For FLAG-tagged mutants (strain JVS-7033 with HP1334– 3xFLAG and JVS-7036 with S1–3xFLAG) a DNA fragment was generated by overlapping of four PCR products (in two steps; for a scheme see Figure 2A). The four PCR products A, B, C and D are: (A) a 500 bp region upstream of the STOP codon of protein X with a part of the 3xFLAG (for HP1334 use JVO-5140/5141 for the PCR with on genomic DNA; for S1 use JVO-5146/5191), (B) the 3xFLAG tag (PCR with JVO-5142/5143 on any template containing the 3xFLAG tag, e.g., pSUB1122 ), (C) the aphA-3′ kanamycin resistance gene45 was amplified with primers JVO5068/HPKterm on genomic DNA of the HPnc5490 deletion strain used in 6 to introduce a cassette containing the promoter of HPnc5490 and a terminator, and (D) a 500 bp region downstream of the STOP codon of protein X (for HP1334 use

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(Table S6-S7). For the 3′MS2-tagged template, DNA containing the T7 promotor upstream of the RNA HPnc6910 and the downstream part of the MS2 tag (PCR with JVO-4761/4771 on genomic DNA of HP26695) was mixed with DNA containing the MS2 tag (PCR with JVO-4773/4774 on any template containing the dimeric MS2 tag, e.g., pRR-0520) in equimolar amounts and overlap PCR with JVO-4202/4773 was performed. For the 5′MS2-tagged template, DNA containing the T7 promotor and the MS2-tag (PCR with JVO-4201/4203 on a template containing the dimeric MS2 tag) and DNA containing part of the MS2 tag and the RNA HPnc6910 (PCR with JVO4762/4765 on genomic DNA of HP26695) were combined in equimolar amounts and overlap PCR with JVO-4202/4762 was performed. In-vitro transcription was then performed with the MEGAscript T7 kit (Ambion) according to the manufacturer instructions. Affinity purification with aptamer-tagged RNA HPnc6910. H. pylori 26995 wild type was grown in liquid culture to an OD600 of ∼1. Cells equivalent to 25 OD600 were chilled on ice for 20 min and harvested (20 min, 2,900 g, 4°C). Cells were resuspended in 1 ml Buffer A (20 mM Tris–HCl pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT) and subsequently centrifuged (5 min, 11 200 g, 4°C). The pellets were shockfrozen in liquid nitrogen and stored at -80°C. Frozen pellets were thawed on ice, resuspended in 0.5 ml Buffer A for each 25 OD600, followed by cell breakage using a French press (French Pressure Cell Press, SLM instruments). A pressure of 800 psi was applied to the cells for three times. Afterwards, the lysate was cleared by centrifugation (20 min, 2,900 g, 4°C) and the soluble fraction was incubated with 300 pmol of in-vitro transcribed RNA for 30 min at 4°C. All steps for affinity purification were performed at 4°C. For preparation of the affinity column, 100 µl amylose resin (New England Biolabs, #E8021S) was applied to Bio-Spin disposable chromatography columns (BioRad, #732–6008). The prepared column was washed three times with 2 ml Buffer A. Next, 600 pmol MS2-MBP coat protein (purification of the protein was done as described in ref. 20) diluted in 1 ml Buffer A was immobilized on the amylose resin, and the column was washed with 2 ml of Buffer A. Subsequently, the mixture of lysate and in-vitro transcribed RNA was loaded onto the column, followed by three washes with 2 ml Buffer A. For RNA analysis, aliquots equivalent to 5 pmol of the lysate, the flow-through and the wash fractions were mixed with 1 ml TRIzol Reagent (Invitrogen), and RNA was isolated and precipitated as described in.47 For protein analysis, aliquots equivalent to 0.5 OD600 were mixed with protein loading buffer (Fermentas; #R0891) to a final volume of 100 µl. Finally, RNA and proteins were eluted from the column with 900 ml of Buffer A containing 12 mM maltose. Eluted RNA was extracted with phenol–chloroform– isoamylalcohol [25:24:1 (v/v), Roth], followed by ethanol (3 vol) precipitation of the aqueous phase. Once re-dissolved in 50 µl water, the RNA was stored at - 20°C. For protein isolation, the organic phase was subjected to acetone precipitation at -20°C over night. The pellet was washed twice with acetone and airdried. The pellet was solved in 100 µl 1x protein loading buffer. The purified RNA was quantified on a Nanodrop Machine

(NanoDrop Technologies). RNA samples were denatured for 3 min at 95°C in protein loading buffer and separated on 6% polyacrylamide/7 M urea gels. Proteins were denatured for 5 min at 96°C, and separated by sodium dodecyl sulfate (SDS)—PAGE (PAGE) using 15% polyacrylamide gels. Co-immunoprecipitation experiments with 3xFLAG tagged HP1334 and S1 protein. FLAG-tagged H. pylori strains were grown in liquid culture to an OD600 of ~2. Cells equivalent to 100 OD600 were chilled on ice for 20 min and harvested (20 min, 2900 g, 4°C). Cells were resuspended in 1 ml Buffer A (20 mM Tris-HCl pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT) and subsequently centrifuged (5 min, 11,200 g, 4°C). The pellets were shock-frozen in liquid nitrogen and stored at -80°C. Frozen pellets were thawed on ice, resuspended in 1.5 ml Buffer A and culture aliquots equivalent to 5 OD600 for RNA and 0.5 OD600 for protein were removed. Cells were broken using a French press as described above and the lysate was cleared by centrifugation (20 min, 2,900 g, 4°C). The soluble fraction was incubated with 35 µl FLAG antibody (Monoclonal ANTI-FLAG M2, Sigma, #F 1804) for 30 min at 4°C, followed by rocking with 75 µl prewashed Protein A sepharose (#P6649, Sigma) for additional 30 min at 4°C. After collecting the beads (1 min, 11 200 g, 4°C), the supernatant was removed and the beads were washed 5 times with 500 µl Buffer A. For RNA analysis, additionally to the culture samples aliquots equivalent to 5 pmol of the lysate, the supernatant and the wash fractions were mixed with 1 ml TRIzol and RNA was isolated and precipitated as described in.47 For protein analysis, aliquots equivalent to 0.5 OD600 were mixed with protein loading buffer to a final volume of 100 µl. Finally, 500 µl Buffer A was added to the beads and RNA and proteins were separated by phenol-chloroform-isoamylalcohol extraction, precipitated and analyzed as described for the affinity purification. The total RNA amounts isolated via co-IP were 730 ng for WT, 990 ng for HP1334-3xFLAG and 2010 ng for S1-3xFLAG. The total amounts are used to calculate the RNA enrichment factor (RNA-E). The RNA-E for HP1334-3xFLAG is 1.36 and results from the ratio of the RNA isolated for HP1334–3xFLAG strain and the RNA isolated for WT strain (= 990 ng/730 ng). The RNA-E for S1-3xFLAG is 2.75 and results from the ratio of the RNA isolated for S1–3xFLAG strain and the RNA isolated for WT strain (= 2010 ng/730 ng). The RNA-E was taken into account for the graphical representation of the read numbers (Figs. 4 and 5A; Fig. S3): y axis was scaled according to the RNA-E. cDNA Library preparation and deep sequencing. For cDNA preparation, co-immunoprecipitated RNA was digested with DNase I. cDNA library preparation and 454 pyrosequencing were performed as previously described,48 but omitting size fractionation. Sequencing was performed on a Roche 454 FLX machine. For mapping of cDNAs, 5′-linker and poly-A-tail clipped reads of at least 18 nt were aligned to the H. pylori 26695 genome (NC_000915) using segemehl.49 For each library, graphs representing the number of mapped reads per nucleotide were calculated and visualized using the Integrated Genome Browser (IGB) version 6.1 software from Affymetrix (genoviz.sourceforge.

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net/) as previously described.9 The read numbers of the different libraries were normalized for graphical representation (all read numbers were normalized to the library with the lowest read number). RNA and protein analysis of mutant strains during growth. H. pylori 26995, JVS-7033 and JVS-7107 were grown in liquid culture and samples between an OD600 of 0.2 and 3 were harvested. For RNA samples, 8 ml cells were mixed with 1 ml STOP-mix (5% phenol, 95% ethanol) and snap-frozen in liquid nitrogen. After thawing on ice, bacteria were pelleted and the RNA was isolated as described in.6 For protein samples, 0.5 OD600 were removed and centrifuged for 2 min at 11,200 g at 4°C. The pellet was resuspended in 100 µl protein loading buffer and denatured for 5 min at 96°C. RNA samples (8 µg each) and protein samples (0.025 OD600 each) were analyzed as described for the affinity purification Northern blot analysis. After PAGE, the RNA was transferred to Hybond-XL membranes (GE Healthcare) by electro-blotting (1 h, 50 V, 4°C) in a tank electro blotter (Peqlab, #52-WEB20), and cross-linked to membrane by exposure to UV light (302 nm) for 4 min. The MS2-tagged RNAs were detected using (gamma-32P) ATP 5′-end-labeled oligodeoxyribonucleotide JVO3562, HPnc6910 using JVO-2711 and 6S RNA using JVO-2136, respectively. Prehybridization and hybridization of membranes with oligonucleotides was performed for 1 h each in RotiHybri-Quick buffer (Roth) at 42°C. Following hybridization, membranes were rinsed in 5x SSC, followed by three washing steps at 42°C for 15 min in SSC (5x, 1x and 0.5x)/0.1% SDS solutions. Signals were visualized on a phosphorimager (Phosphorimager, LA-3000 Series, Fuji), and band intensities were quantified with the AIDA software (Raytest, Germany). Western blot analysis and Coomassie/silver staining. For western blot analysis, polyvinylidene difluoride membrane (PVDF, PerkinElmer) was activated by incubation in methanol (90 sec), H2O (5 min) and transfer buffer (5 min), respectively.

After SDS–PAGE, the gel was blotted for 2 h at 2 mA/cm2 and 4°C in a semi-dry electro blotter (Peqlab, #52–2020) onto the PVDF membrane in transfer buffer. After rinsing in TBST20, the membrane was incubated over night in 10% dry milk in TBST20. FLAG-tagged proteins were detected using a monoclonal anti-FLAG antibody (Sigma-Aldrich, #F2555, 1:1000 in TBST20-BSA), for 1 h at room temperature under agitation. Membranes were washed 6x5 min in TBST20, incubated with either anti-mouse–horseradish peroxidase (GE Healthcare, 1:5000 in TBST20-BSA) for 1 h at room temperature and washed again 6x10 min in TBST20. Blots were analyzed using Western Lightning Reagent (Perkin Elmer), and signals detected with a Fuji LAS-3000 CCD camera. Silver staining of the 15% polyacrylamide gel was performed according to.50 Page Blue (Fermentas) was used for Coomassie staining according to the manfacturer’s instructions. Disclosure of Potential Conflicts of Interest

© 2012 Landes Bioscience. No potential conflicts of interest were disclosed. Acknowledgments

This work was funded by the Federal Ministry of Education, Science, Research and Technology (BMBF NGFN: 01GS08200 to JV and RR), and the German Research Council Priority Programme SPP1258 Sensory and regulatory RNAs in bacteria (grant Vo857/4–2 to JV). Renate Rieder was supported by an Erwin-Schrödinger Fellowship (J 2949-B12) of the Austrian Science Fund FWF. We thank Konrad U. Förstner for help with the analysis of deep sequencing data.

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Supplemental material

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