R-loop Structures Research Articles

Interplay between DNA sequence and negative superhelicity drives R-loop structures

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

SFPQ and NONO suppress RNA:DNA-hybrid-related telomere instability

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding proteins NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telomeres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids.

DNA Topoisomerase I differentially modulates R-loops across the human genome

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.ResultsHere, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.ConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.

R-Loops in Motor Neuron Diseases.

R loops are transient three-stranded nucleic acid structures that form physiologically during transcription when a nascent RNA transcript hybridizes with the DNA template strand, leaving a single strand of displaced nontemplate DNA. However, aberrant persistence of R-loops can cause DNA damage by inducing genomic instability. Indeed, evidence has emerged that R-loops might represent a key element in the pathogenesis of human diseases, including cancer, neurodegeneration, and motor neuron disorders. Mutations in genes directly involved in R-loop biology, such as SETX (senataxin), or unstable DNA expansion eliciting R-loop generation, such as C9ORF72 HRE, can cause DNA damage and ultimately result in motor neuron cell death. In this review, we discuss current advancements in this field with a specific focus on motor neuron diseases associated with deregulation of R-loop structures. These mechanisms can represent novel therapeutic targets for these devastating, incurable diseases.

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination

SummaryClass switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Abstract B188: DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity

Abstract DNA topoisomerases have an important function in preventing replication/transcription (R/T) conflicts caused by RNA polymerases elongating in the opposite (or same) direction of advancing replication fork. Published data demonstrated that such persistent R/T conflicts can be rescued by overexpression of RNase H1, which specifically degrades the RNA of DNA:RNA hybrids present in R-loop structures. R loops are a common non-B DNA structure of cellular genomes that mainly occur during transcription. R loops can lead to genome instability; however, they also play a role in important cellular functions. Here, we have determined the dynamics of cellular R loops generated by chemical poisoning of DNA topoisomerases (TOP1 and TOP2) and the role of the proteasome in DNA damage induction by performing detailed kinetics analyses of cellular R loops by immunofluorescence microscopy. Treatment of HeLa and U2OS cells with clinically used topoisomerase inhibitors (camptothecin, LMP-776, MJ-III-65, doxorubicin, sabarubicin) triggers an immediate increase of nucleoplasmic R loops from 2 to 10-20 minutes of treatment, followed by a dramatic reduction of R-loop levels at 1 hour. Concomitantly with the reduction of R-loop structures, DNA damage was detected as phosphorylation of histone H2AX, a marker of DNA double-stranded breaks (DSB). Pretreating cells with MG132 (a proteasome inhibitor) stabilized the presence of R loops at longer treatment times and concomitantly reduced DSB formation, suggesting that the degradation of poisoned topoisomerases is necessary to trigger the pathway resulting in DSB formation. Overexpression of RNaseH1 in U2OS cells abolishes the transient increase of R loops and the induction of DNA damage. Topoisomerase poisoning is able to effectively kill the studied human cancer cells. However, overexpression of RNaseH1 or MG132 markedly reduced cell killing induced by R/T conflicts generated by topoisomerase inhibitors. The results are overall consistent with a necessary function of nuclear proteasome in generating a free DNA break from abortive topoisomerase cleavage complexes that will eventually lead to DSBs. Citation Format: Jessica Marinello, Maria Delcuratolo, Yves Pommier, Monica Binaschi, Andrea Pellacani, Giovanni Capranico. DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B188.

Hybrids in the chromatin.

New genomic maps reveal that R-loop structures formed upon hybridization of nascent RNA transcripts to the DNA template are a common characteristic of Arabidopsis chromatin that may have a broad impact on gene expression.

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv) of S9.6 showed high affinity (0.6 nM) for DNA—RNA and also a high affinity (2.7 nM) for RNA—RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

Ataxin-2: From RNA Control to Human Health and Disease.

RNA-binding proteins play fundamental roles in the regulation of molecular processes critical to cellular and organismal homeostasis. Recent studies have identified the RNA-binding protein Ataxin-2 as a genetic determinant or risk factor for various diseases including spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS), amongst others. Here, we first discuss the increasingly wide-ranging molecular functions of Ataxin-2, from the regulation of RNA stability and translation to the repression of deleterious accumulation of the RNA-DNA hybrid-harbouring R-loop structures. We also highlight the broader physiological roles of Ataxin-2 such as in the regulation of cellular metabolism and circadian rhythms. Finally, we discuss insight from clinically focused studies to shed light on the impact of molecular and physiological roles of Ataxin-2 in various human diseases. We anticipate that deciphering the fundamental functions of Ataxin-2 will uncover unique approaches to help cure or control debilitating and lethal human diseases.

DNA Topoisomerase 1 Prevents R-loop Accumulation to Modulate Auxin-Regulated Root Development in Rice

R-loop structures (RNA:DNA hybrids) have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA topoisomerase 1 (TOP1), an essential manipulator of DNA topology during RNA transcription and DNA replication processes, can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription. TOP1 is required for plant development, but little is known about its function in preventing co-transcriptional R-loop accumulation in various biological processes in plants. Here we show that knockdown of OsTOP1 strongly affects rice development, causing defects in root architecture and gravitropism, which are the consequences of misregulation of auxin signaling and transporter genes. We found that R-loops are naturally formed at rice auxin-related gene loci, and overaccumulate when OsTOP1 is knocked down or OsTOP1 protein activity is inhibited. OsTOP1 therefore sets the accurate expression levels of auxin-related genes by preventing the overaccumulation of inherent R-loops. Our data reveal R-loops as important factors in polar auxin transport and plant root development, and highlight that OsTOP1 functions as a key to link transcriptional R-loops with plant hormone signaling, provide new insights into transcriptional regulation of hormone signaling in plants.

Type II and type V CRISPR effector nucleases from a structural biologist’s perspective

The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are "universal" DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5'- or 3'-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases.

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals

R-loops are three-stranded nucleic acid structures formed upon annealing of an RNA strand to one strand of duplex DNA. We profiled R-loops using a high-resolution, strand-specific methodology in human and mouse cell types. R-loops are prevalent, collectively occupying up to 5% of mammalian genomes. R-loop formation occurs over conserved genic hotspots such as promoter and terminator regions of poly(A)-dependent genes. In most cases, R-loops occur co-transcriptionally and undergo dynamic turnover. Detailed epigenomic profiling revealed that R-loops associate with specific chromatin signatures. At promoters, R-loops associate with a hyper-accessible state characteristic of unmethylated CpG island promoters. By contrast, terminal R-loops associate with an enhancer- and insulator-like state and define a broad class of transcription terminators. Together, this suggests that the retention of nascent RNA transcripts at their site of expression represents an abundant, dynamic, and programmed component of the mammalian chromatin that affects chromatin patterning and the control of gene expression.

Editorial: DNA and RNA Metabolism Meet at Chromatin to Control Genome Stability.

Editorial: DNA and RNA Metabolism Meet at Chromatin to Control Genome Stability.

Disease-associated repeat instability and mismatch repair.

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

ScPif1 DNA helicase is the prototypical member of a 5′-to-3′ helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5′-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA–RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.

RNA Exosome-Regulated Long Non-Coding RNA Transcription Controls Super-Enhancer Activity

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

Friedreich's ataxia – a case of aberrant transcription termination?

Reduced expression of the mitochondrial protein Frataxin (FXN) is the underlying cause of Friedreich's ataxia. We propose a model of premature termination of FXN transcription induced by pathogenic expanded GAA repeats that links R-loop structures, antisense transcription, and heterochromatin formation as a novel mechanism of transcriptional repression in Friedreich's ataxia.

BRCA1 Recruitment to Transcriptional Pause Sites Is Required for R-Loop-Driven DNA Damage Repair

SummaryThe mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

Out of balance: R-loops in human disease.

R-loops are cellular structures composed of an RNA/DNA hybrid, which is formed when the RNA hybridises to a complementary DNA strand and a displaced single-stranded DNA. R-loops have been detected in various organisms from bacteria to mammals and play crucial roles in regulating gene expression, DNA and histone modifications, immunoglobulin class switch recombination, DNA replication, and genome stability. Recent evidence suggests that R-loops are also involved in molecular mechanisms of neurological diseases and cancer. In addition, mutations in factors implicated in R-loop biology, such as RNase H and SETX (senataxin), lead to devastating human neurodegenerative disorders, highlighting the importance of correctly regulating the level of R-loops in human cells. In this review we summarise current advances in this field, with a particular focus on diseases associated with dysregulation of R-loop structures. We also discuss potential therapeutic approaches for such diseases and highlight future research directions.