sRNA-Mediated Control of Transcription Termination in E. coli.

Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli.

In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.

iRAPs curb antisense transcription in E. coli.

RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.

In vivo expression technology and 5' end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection.

Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5' end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Temperature-dependent sRNA transcriptome of the Lyme disease spirochete.

BACKGROUND: Transmission of Borrelia burgdorferi from its tick vector to a vertebrate host requires extensive reprogramming of gene expression. Small regulatory RNAs (sRNA) have emerged in the last decade as important regulators of bacterial gene expression. Despite the widespread observation of sRNA-mediated gene regulation, only one sRNA has been characterized in the Lyme disease spirochete B. burgdorferi. We employed an sRNA-specific deep-sequencing approach to identify the small RNA transcriptome of B. burgdorferi at both 23 °C and 37 °C, which mimics in vitro the transmission from the tick vector to the mammalian host. RESULTS: We identified over 1000 sRNAs in B. burgdorferi revealing large amounts of antisense and intragenic sRNAs, as well as characteristic intergenic and 5' UTR-associated sRNAs. A large fraction of the novel sRNAs (43%) are temperature-dependent and differentially expressed at the two temperatures, suggesting a role in gene regulation for adaptation during transmission. In addition, many genes important for maintenance of Borrelia during its enzootic cycle are associated with antisense RNAs or 5' UTR sRNAs. RNA-seq data were validated for twenty-two of the sRNAs via Northern blot analyses. CONCLUSIONS: Our study demonstrates that sRNAs are abundant and differentially expressed by environmental conditions suggesting that gene regulation via sRNAs is a common mechanism utilized in B. burgdorferi. In addition, the identification of antisense and intragenic sRNAs impacts the broadly used loss-of-function genetic approach used to study gene function and increases the coding potential of a small genome. To facilitate access to the analyzed RNA-seq data we have set-up a website at http://www.cibiv.at/~niko/bbdb/ that includes a UCSC browser track hub. By clicking on the respective link, researchers can interactively inspect the data in the UCSC genome browser (Kent et al., Genome Res 12:996-1006, 2002).

The double-stranded transcriptome of Escherichia coli.

Advances in high-throughput transcriptome analyses have revealed hundreds of antisense RNAs (asRNAs) for many bacteria, although few have been characterized, and the number of functional asRNAs remains unknown. We have developed a genome-wide high-throughput method to identify functional asRNAs in vivo. Most mechanisms of gene regulation via asRNAs require an RNA-RNA interaction with its target RNA, and we hypothesized that a functional asRNA would be found in a double strand (dsRNA), duplexed with its cognate RNA in a single cell. We developed a method of isolating dsRNAs from total RNA by immunoprecipitation with a ds-RNA specific antibody. Total RNA and immunoprecipitated dsRNA from Escherichia coli RNase III WT and mutant strains were deep-sequenced. A statistical model was applied to filter for biologically relevant dsRNA regions, which were subsequently categorized by location relative to annotated genes. A total of 316 potentially functional asRNAs were identified in the RNase III mutant strain and are encoded primarily opposite to the 5' ends of transcripts, but are also found opposite ncRNAs, gene junctions, and the 3' ends. A total of 21 sense/antisense RNA pairs identified in dsRNAs were confirmed by Northern blot analyses. Most of the RNA steady-state levels were higher or detectable only in the RNase III mutant strain. Taken together, our data indicate that a significant amount of dsRNA is formed in the cell, that RNase III degrades or processes these dsRNAs, and that dsRNA plays a major role in gene regulation in E. coli.

Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents.

Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq's activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq's strand displacement as well as its annealing activity are strongly dependent on the substrate's GC-content. However, this is due to Hfq's preferred binding of AU-rich sequences and not to the substrate's thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq's strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq's thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.

Revisiting the coding potential of the E. coli genome through Hfq co-immunoprecipitation.

Hfq is a global regulator of gene expression in bacteria undergoing adaptation to changing environmental conditions. Its major function is to promote RNA-RNA interactions between regulatory small RNAs (sRNAs) and their target mRNAs. Previously, we demonstrated that Hfq binds many antisense RNAs (asRNAs) in vitro and hypothesized that Hfq may play a role in regulating gene expression via asRNAs. To investigate the E. coli Hfq-binding transcriptome in more detail, we co-immunoprecipitated and deep-sequenced RNAs bound to Hfq in vivo. We detected many new Hfq-binding sRNAs and observed that almost 300 mRNAs bind to Hfq. Among these, several are known to be sRNA targets. We identified 25 novel RNAs, which are transcribed from within protein coding regions and named them intragenic RNAs (intraRNAs). Furthermore, 67 asRNAs were co-immunoprecipitated with Hfq, demonstrating that Hfq binds antisense transcripts in vivo. Northern blot analyses confirmed the deep-sequencing results and demonstrated that many of the novel Hfq-binding RNAs identified are regulated by Hfq.

Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA.

CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.

The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation.

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

Genomic SELEX: a discovery tool for genomic aptamers.

Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.

Screening for engineered neomycin riboswitches that control translation initiation.

Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.

Mechanisms of StpA-mediated RNA remodeling.

In bacteria, transcription, translation and gene regulation are highly coupled processes. The achievement of a certain functional structure at a distinct temporal and spatial position is therefore essential for RNA molecules. Proteins that facilitate this proper folding of RNA molecules are called RNA chaperones. Here a prominent example from E. coli is reviewed: the nucleoid associated protein StpA. Based on its various RNA remodeling functions, we propose a mechanistic model that explains how StpA promotes RNA folding. Through transient interactions via the RNA backbone, thereby shielding repelling charges in RNA, it pre-positions the RNA molecules for the successful formation of transition states from encounter complexes.

The C-terminal domain of Escherichia coli Hfq is required for regulation.

The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the gamma- and beta-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq(65)), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

RNA polymerase II-binding aptamers in human ACRO1 satellites disrupt transcription in cis.

Transcription elongation is a highly regulated process affected by many proteins, RNAs and the underlying DNA. Here we show that the nascent RNA can interfere with transcription in human cells, extending our previous findings from bacteria and yeast. We identified a variety of Pol II-binding aptamers (RAPs), prominent in repeat elements such as ACRO1 satellites, LINE1 retrotransposons and CA simple repeats, and also in several protein-coding genes. ACRO1 repeat, when translated in silico, exhibits ~50% identity with the Pol II CTD sequence. Taken together with a recent proposal that proteins in general tend to interact with RNAs similar to their cognate mRNAs, this suggests a mechanism for RAP binding. Using a reporter construct, we show that ACRO1 potently inhibits Pol II elongation in cis. We propose a novel mode of transcriptional regulation in humans, in which the nascent RNA binds Pol II to silence its own expression.

In vitro selection of RNA aptamers derived from a genomic human library against the TAR RNA element of HIV-1.

The transactivating responsive (TAR) element is a RNA hairpin located in the 5' untranslated region of HIV-1 mRNA. It is essential for full-length transcription of the retroviral genome and therefore for HIV-1 replication. Hairpin aptamers that generate highly stable and specific complexes with TAR were previously identified, thus decreasing the level of TAR-dependent expression in cultured cells [Kolb, G., et al. (2006) RNA Biol. 3, 150-156]. We performed genomic SELEX against TAR using a human RNA library to identify human transcripts that might interact with the retroviral genome through loop-loop interactions and potentially contribute to the regulation of TAR-mediated processes. We identified a genomic aptamer termed a1 that folds as a hairpin with an apical loop complementary to five nucleotides of the TAR hexanucleotide loop. Surface plasmon resonance experiments performed on a truncated or mutated version of the a1 aptamer, in the presence of the Rop protein of Escherichia coli, indicate the formation of a highly stable a1-TAR kissing complex. The 5' ACCCAG loop of a1 constitutes a new motif of interaction with the TAR loop.

Dissecting RNA chaperone activity.

Many RNA-binding proteins help RNAs to fold via their RNA chaperone activity. This term has been used widely without accounting for the diversity of the observed reactions, which include complex events like restructuring of misfolded catalytic RNAs, promoting the assembly of RNA-protein complexes, and mediating RNA-RNA interactions. Proteins display very diverse activities depending on the assays used to measure RNA chaperone activity. To classify proteins with this activity, we compared three exemplary proteins from E. coli, host factor Hfq, ribosomal protein S1, and the histone-like protein StpA for their abilities to promote two simple reactions, RNA annealing and strand displacement. The results of a FRET-based assay show that S1 promotes only RNA strand displacement while Hfq solely enhances RNA annealing. StpA, in contrast, is active in both reactions. To test whether the two activities can be assigned to different domains of the bipartite-structured StpA, we assayed the purified N- and C- terminal domains separately. While both domains are unable to promote RNA annealing, we can attribute the RNA strand displacement activity of StpA to the C-terminal domain. Correlating with their RNA annealing activities, only Hfq and full-length StpA display simultaneous binding of two RNAs, suggesting a matchmaker-like model for this activity. For StpA, this "RNA crowding" requires protein-protein interactions, since a dimerization-deficient StpA mutant lost the ability to bind and anneal two RNAs. These results underline the difference between the two reaction types, making it necessary to distinguish and classify proteins according to their specific RNA chaperone activities.