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R-loops are nucleic acid structures composed of a DNA:RNA hybrid with a displaced non-template single-stranded DNA. Current approaches to identify and map R-loop formation across the genome employ either an antibody targeted against R-loops (S9.6) or a catalytically inactivated form of RNase H1 (dRNH1), a nuclease that can bind and resolve DNA:RNA hybrids via RNA exonuclease activity. This overview article outlines several ways to map R-loops using either methodology, explaining the differences and similarities among the approaches. Bioinformatic analysis of R-loops involves several layers of quality control and processing before visualizing the data. This article provides resources and tools that can be used to accurately process R-loop mapping data and explains the advantages and disadvantages of the resources as compared to one another. © 2024 Wiley Periodicals LLC.

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R-loops are regulators of many cellular processes and are threats to genome integrity. Therefore, understanding the mechanisms underlying the regulation of R-loops is important. Inspired by the findings on RNase H1-mediated R-loop degradation or accumulation, we focused our interest on the regulation of RNase H1 expression. In the present study, we report that G9a positively regulates RNase H1 expression to boost R-loop degradation. CHCHD2 acts as a repressive transcription factor that inhibits the expression of RNase H1 to promote R-loop accumulation. Sirt1 interacts with CHCHD2 and deacetylates it, which functions as a corepressor that suppresses the expression of downstream target gene RNase H1. We also found that G9a methylated the promoter of RNase H1, inhibiting the binding of CHCHD2 and Sirt1. In contrast, when G9a was knocked down, recruitment of CHCHD2 and Sirt1 to the RNase H1 promoter increased, which co-inhibited RNase H1 transcription. Furthermore, knockdown of Sirt1 led to binding of G9a to the RNase H1 promoter. In summary, we demonstrated that G9a regulates RNase H1 expression to maintain the steady-state balance of R-loops by suppressing the recruitment of CHCHD2/Sirt1 corepressors to the target gene promoter.

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Here, we describe an assay that enables mapping of 5'-ends across the genome using next-generation sequencing on an Illumina platform, 5'-End-sequencing (5'-End-seq). We use this method to map free 5'-ends in mtDNA isolated from fibroblasts. This method can be used to answer key questions regarding DNA integrity, DNA replication mechanisms and to identify priming events, primer processing, nick processing, and double strand break processing on the entire genome.

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Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

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RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

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R loops are abundant noncanonical DNA-RNA hybrid structures that can occur during DNA-based processes, such as transcription, replication and DNA damage, and can lead both to physiologically favorable and pathological outcomes. With an increasing body of work feeding the field of R loop biology, our understanding of the processes in which R loops intervene and the consequences of meddling with R loop formation and dissolution has greatly increased but it has also led to new questions and sometimes opposing possibilities. Proper detection of these structures is a crucial factor to advance our knowledge about R loops and factors associated with their formation and removal. Here, we describe the use of fluorescently tagged HBD, the hybrid-binding domain of RNase H1, as a tool for analyzing DNA-RNA hybrids in different contexts using live-cell microscopy and immunofluorescence experiments.

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RNase H1 has become an essential tool to uncover the physiological and pathological roles of R-loops, three-stranded structures consisting of and RNA-DNA hybrid opposite to a single DNA strand (ssDNA). RNase H1 degrades the RNA portion of the R-loops returning the two DNA strands to double-stranded form (dsDNA). Overexpression of RNase H1 in different systems has helped to address the questions of where R-loops are located, their abundance, and mechanisms of formation, stability, and degradation. In this chapter we review multiple studies that used RNase H1 as an instrument to investigate R-loops multiple functions and their relevance in health and diseases.

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By temporarily distorting the DNA double helix, the moving RNA polymerases can lead to the formation of non-B DNA structures. One of the most abundant and largest non-B DNA structures in the genome is the R-loop, a three-stranded structure forming when the nascent RNA hybridizes with its DNA template, thereby extruding the non-template DNA strand. Growing evidence suggests that at least a subset of R-loops could induce transcription stress and genome instability, although the direct, primary consequences of R-loop formation on the surrounding chromatin are still unclear.To understand the direct impact of R-loops on transcription and genome stability, accurate and quantitative mapping of R-loops is essential. R-loop mapping is commonly achieved using the antibody-based DNA:RNA Immunoprecipitation (DRIP) strategy. While it is reasonably straightforward to obtain robust DRIP enrichments from human cells, this has proved harder in yeast, where DRIP signals are often relatively weak, with a poor signal-to-noise ratio. Although it is unclear whether such weak signals stem from a technical or a biological reality, they make the accurate quantification of DRIP signals all the more important, especially when deep sequencing is used to monitor and quantify the distribution of R-loops genome-wide. Here we propose a DRIP protocol that has been optimized for the mapping and the quantification of R-loops in Schizosaccharomyces pombe but that can also be used in Saccharomyces cerevisiae. As a result, this protocol can be used to generate calibrated DRIP-seq data, where genomic DNA extracted from S. cerevisiae serves as spike-in reference.

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Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection.

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R-loops, consisting of a DNA:RNA hybrid and a single-stranded DNA (ssDNA), form naturally as functional chromosome structures and are crucial in many vital biological processes. However, disrupted R-loop homeostasis will threat to the integrity and stability of genome. As the endonuclease, RNase H1 can efficiently recognize and remove excess R-loops to protect organisms from DNA damage induced by R-loop over-accumulation. Here, we investigated the function of RNase H1 in Physcomitrium (Physcomitrella) patens to illustrate its important role in the evolution of plants. We found that PpRNH1A dysfunction seriously affected shoot growth and branch formation in P. patens, revealing a noticeable functional difference between PpRNH1A and AtRNH1A of Arabidopsis. Furthermore, auxin signaling was significantly affected at the transcriptional level in PpRNH1A mutant plants, as a result of the accumulation of R-loops at several auxin-related genes. This study provides evidence that PpRNH1A regulates the development of P. patens by controlling R-loop formation at specific loci to modulate the transcription of auxin-related genes. It also highlights the interspecific functional differences between early land plants and vascular plants, despite crucial and conserved role of RNase H1 played in maintaining R-loop homeostasis.

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Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

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Circular intronic RNAs (ciRNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from pre-mRNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ciRNAs in human cells. Many ciRNAs contain high GC% and tend to form DNA:RNA hybrids (R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ciRNA-producing loci. One ciRNA, ciankrd52, shows a stronger ability of R-loop formation than that of its cognate pre-mRNA by maintaining a locally open RNA structure in vitro. This allows the release of pre-mRNA from R-loops by ci-ankrd52 replacement and subsequent ciRNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ciRNA degradation likely represents a mechanism that on one hand limits ciRNA accumulation by recruiting RNase H1 and on the other hand resolves R-loops for transcriptional elongation at some GC-rich ciRNA-producing loci.

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The ribosomal gene DNA (rDNA) often forms secondary constrictions in the chromosome; however, the molecular mechanism involved remains poorly understood. Here, we report that occurrence of rDNA constriction was increased in the chromosomes in human cancer cell lines compared with normal cells and that decondensed rDNA was significantly enhanced after partial inhibition of rDNA transcription. rDNA transcription was found during the S phase when replication occurred, and thus, DNA replication inhibitors caused constriction formation through hindering rDNA transcription. Inhibition of ataxia ATR (telangiectasia-mutated and RAD3-related) induced rDNA constriction formation. Replication stress or transcription inhibition increased R-loop formation. Topoisomerase I and RNase H1 suppressed secondary constriction formation. These data demonstrate that transcription stress causes the accumulation of stable R-loops (RNA-DNA hybrid) and subsequent constriction formation in the chromosomes.

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Research and drug development concerning rare diseases are at the cutting edge of scientific technology. To date, over 7,000 rare diseases have been identified. Despite their individual rarity, 1 in 10 individuals worldwide is affected by a rare condition. For the majority of these diseases, there is no treatment, much less cure; therefore, there is an urgent need for new therapies to extend and improve quality of life for persons who suffer from them. Here we focus specifically on rare neuromuscular diseases. Currently, genetic medicines using short antisense oligonucleotides (ASO) or small interfering ribonucleic acids that target RNA transcripts are achieving spectacular success in treating these diseases. For Duchenne muscular dystrophy (DMD), the state-of-the-art is an exon skipping therapy using an antisense oligonucleotide, which is prototypical of advanced precision medicines. Very recently, golodirsen and viltolarsen, for treatment of DMD patients amenable to skipping exon 53, have been approved by regulatory agencies in the USA and Japan, respectively. Here, we review scientific and clinical progress in developing new oligonucleotide therapeutics for selected rare neuromuscular diseases, discussing their efficacy and limitations.

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The c.151C>T founder mutation in COCH is a frequent cause of late-onset, dominantly inherited hearing impairment and vestibular dysfunction (DFNA9) in the Dutch/Belgian population. The initial clinical symptoms only manifest between the 3rd and 5th decade of life, which leaves ample time for therapeutic intervention. The dominant inheritance pattern and established non-haploinsufficiency disease mechanism indicate that suppressing translation of mutant COCH transcripts has high therapeutic potential. Single-molecule real-time (SMRT) sequencing resulted in the identification of 11 variants with a low population frequency (<10%) that are specific to the c.151C>T mutant COCH allele. Proof of concept was obtained that gapmer antisense oligonucleotides (AONs), directed against the c.151C>T mutation or mutant allele-specific intronic variants, are able to induce mutant COCH transcript degradation when delivered to transgenic cells expressing COCH minigenes. The most potent AON, directed against the c.151C>T mutation, was able to induce a 60% decrease in mutant COCH transcripts without affecting wild-type COCH transcript levels. Allele specificity decreased when increasing concentrations of AON were delivered to the cells. With the proven safety of AONs in humans, and rapid advancements in inner ear drug delivery, our in vitro studies indicate that AONs offer a promising treatment modality for DFNA9.

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Antisense technology is beginning to deliver on the broad promise of the technology. Ten RNA-targeted drugs including eight single-strand antisense drugs (ASOs) and two double-strand ASOs (siRNAs) have now been approved for commercial use, and the ASOs in phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are used in cardiovascular outcome studies and several others in very large trials. Interest in the technology continues to grow, and the field has been subject to a significant number of reviews. In this review, we focus on the molecular events that result in the effects observed and use recent clinical results involving several different ASOs to exemplify specific molecular mechanisms and specific issues. We conclude with the prospective on the technology.

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R-loops represent an abundant class of large non-B DNA structures in genomes. Even though they form transiently and at modest frequencies, interfering with R-loop formation or dissolution has significant impacts on genome stability. Addressing the mechanism(s) of R-loop-mediated genome destabilization requires a precise characterization of their distribution in genomes. A number of independent methods have been developed to visualize and map R-loops, but their results are at times discordant, leading to confusion. Here, we review the main existing methodologies for R-loop mapping and assess their limitations as well as the robustness of existing datasets. We offer a set of best practices to improve the reproducibility of maps, hoping that such guidelines could be useful for authors and referees alike. Finally, we propose a possible resolution for the apparent contradictions in R-loop mapping outcomes between antibody-based and RNase H1-based mapping approaches.

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Hereditary transthyretin amyloidosis (hATTR) is a rare autosomal dominant condition in which mutations in the transthyretin gene cause amyloid fibrils to develop and deposit into tissues, affecting primarily the nerves and heart causing polyneuropathy and cardiomyopathy respectively. Standard treatment has been liver transplants to try and eliminate the mutated transthyretin products as the liver is the main source of transthyretin production. A new drug named inotersen (brand name Tagsedi), also known as IONIS-TTRRX, has been approved by the United States Food and Drug Agency, Health Canada, and European Commission in 2018, and introduced to the market for patients in stage 1 and stage 2 hATTR polyneuropathy. Inotersen is a second-generation antisense oligonucleotide with 2'-O-methoxyethyl modification designed to bind to the 3' untranslated region of the transthyretin mRNA in the nucleus of the liver cells. By doing so, it prevents the production of the mutant and wild-type forms of transthyretin, impeding the progression of the disease. In this article, the mechanism of action and safety profile of inotersen will be discussed along with some future directions following its approval.

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In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA. Intriguingly, we also observed that the same ASO induced an accumulation of pre-5S RNA, which may contribute to reduced levels of mature 5S rRNA. As expected, ASO mediated reduction of 5S RNA, and modest inhibition of processing of pre-5S RNA resulted in nucleolar toxicity. However, the toxicity induced was minimal compared with actinomycin D, consistent with its modest effects on pre-5S rRNA. Mechanistically, we show that the accumulation of pre-5S rRNA required ASO hybridization to the cognate rRNA sequence but was independent of RNaseH1 activity. We found that Ro60 and La, proteins known to bind misprocessed RNAs, likely sequester the ASO-pre-5S rRNA species and block RNaseH1 activity, thus identifying another example of competitive mechanisms mediated by proteins that compete with RNaseH1 for binding to ASO-RNA heteroduplexes.

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Preventing transcription-replication head-on conflict (HO-TRC)-triggered R-loop formation is essential for maintaining genome integrity in bacteria, plants, and mammals. The R-loop eraser RNase H can efficiently relax HO-TRCs. However, it is not clear how organisms resist HO-TRC-triggered R-loops when RNase H proteins are deficient. By screening factors that may relieve R-loop accumulation in the Arabidopsis atrnh1c mutant, we find that overexpression of the R-loop helicase RHON1 can rescue the defects of aberrantly accumulated HO-TRC-triggered R-loops co-transcriptionally. In addition, we find that RHON1 interacts with and orchestrates the transcriptional activity of plastid-encoded RNA polymerases to release the conflicts between transcription and replication. Our study illustrates that organisms employ multiple mechanisms to escape HO-TRC-triggered R-loop accumulation and thus maintain genome integrity.

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