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.

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.

Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed “RAPs”. RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.

Alternative splicing is one of the major regulators of both, transcriptome diversity and individual isoform abundance. Therefore regulation of alternative splicing is crucial and yet, due to the complexity of the human genome (23.000 genes, most of which can be alternatively spliced) diverse and multileveled. Identifying and understanding the scope and variety of splicing events is still an ongoing process. We established a novel pipeline to extract splicing events from specific RNA-sequencing datasets and identified numerous splicing events that did not span the entirety of the respective annotated intron but used intronic splice sites. These splicing events could be generally categorized into three groups: 5’recursive (using the exonic splice donor and intronic acceptor), 3’recursive (using an intronic splice donor and an exonic acceptor) and nested (using two intronic splice sites). Surprisingly, the splicing events we found occurred in introns of all lengths, but generally followed the abundance scheme of all introns, i.e. most were found in introns between 500 and 5000 bps. After confirmation of these splice events by conventional methodologies, we further analyzed the impact of intrasplicing on full intron removal. For this we established a luciferase-based reporter which showed that these intronic splicing steps can be beneficial, deleterious or neutral for full intron removal. Thus intrasplicing events can be crucial for determining the transcriptional output. This in part confirms recent findings on recursive splicing events in humans and other vertebrates and further uncovers an additional level of transcriptome regulation based on a yet undiscovered level of flexibility and regulation of splice site selection and its impact on gene expression.

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.

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.

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.

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 sigma(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.

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.

It is a great honor for me as the Editor in Chief of RNA Biology to host this very special commemoration for Carl Woese.The idea for this special Carl Woese issue came during the 18th annual meetin...

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.

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.

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.