Epitranscriptome in action: RNA modifications in the DNA damage response.

Complex pathways involving the DNA damage response (DDR) contend with cell-intrinsic and -extrinsic sources of DNA damage. DDR mis-regulation results in genome instability that can contribute to aging and diseases including cancer and neurodegeneration. Recent studies have highlighted key roles for several RNA species in the DDR, including short RNAs and RNA/DNA hybrids (R-loops) at DNA break sites, all contributing to efficient DNA repair. RNAs can undergo more than 170 distinct chemical modifications. These RNA modifications have emerged as key orchestrators of the DDR. Here, we highlight the function of enzyme- and non-enzyme-induced RNA modifications in the DDR, with particular emphasis on m6A, m5C, and RNA editing. We also discuss stress-induced RNA damage, including RNA alkylation/oxidation, RNA-protein crosslinks, and UV-induced RNA damage. Uncovering molecular mechanisms that underpin the contribution of RNA modifications to DDR and genome stability will have direct application to disease and approaches for therapeutic intervention.

Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS-R-loop-H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis.

SOSS-INTAC: a new gatekeeper of genomic integrity at the interface of transcription and R-loops.

A recent Nature paper by Xu et al.1 describes an important link between RNA polymerase II promoter-proximal pausing and genome stability orchestrated by liquid droplet formation to reduce unwanted R-loop accumulation.

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

What repeat expansion disorders can teach us about the Central Dogma.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.

R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors.

R-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important biological roles. However, perturbation of R-loop levels represents a source of DNA damage and genome instability resulting in human diseases, including cancer and neurodegeneration. In this chapter, we describe a protocol which allows detection of R-loop interactors using affinity purification with S9.6 antibody, specific for RNA/DNA hybrids, followed by Western blotting or mass spectrometry. Multiple specificity controls including addition of synthetic competitors and RNase H treatment are described to verify the specificity of identified R-loop-binding factors. The identification of new R-loop interacting factors and the characterization of their involvement in R-loop biology provides a powerful resource to study the role of these important structures in health and disease.

RNase H2, mutated in Aicardi-Goutières syndrome, resolves co-transcriptional R-loops to prevent DNA breaks and inflammation.

RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières syndrome astrocytes.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.

Transcription-dependent DNA double-strand breaks and human disease.

Accumulation of DNA damage in resting cells is an emerging cause of human disease. We identified a mechanism of DNA double-strand break (DSB) formation in non-replicating cells, which strictly depends on transcription. These transcriptional DSBs arise from the twinned processing of R-loops and topoisomerase I and may underlie neurological disorders and cancers.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks.

Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) is limited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease.

Large XPF-dependent deletions following misrepair of a DNA double strand break are prevented by the RNA:DNA helicase Senataxin.

Deletions and chromosome re-arrangements are common features of cancer cells. We have established a new two-component system reporting on epigenetic silencing or deletion of an actively transcribed gene adjacent to a double-strand break (DSB). Unexpectedly, we find that a targeted DSB results in a minority (<10%) misrepair event of kilobase deletions encompassing the DSB site and transcribed gene. Deletions are reduced upon RNaseH1 over-expression and increased after knockdown of the DNA:RNA helicase Senataxin, implicating a role for DNA:RNA hybrids. We further demonstrate that the majority of these large deletions are dependent on the 3' flap endonuclease XPF. DNA:RNA hybrids were detected by DNA:RNA immunoprecipitation in our system after DSB generation. These hybrids were reduced by RNaseH1 over-expression and increased by Senataxin knock-down, consistent with a role in deletions. Overall, these data are consistent with DNA:RNA hybrid generation at the site of a DSB, mis-processing of which results in genome instability in the form of large deletions.

Senataxin: Genome Guardian at the Interface of Transcription and Neurodegeneration.

R-loops comprise an RNA/DNA hybrid and a displaced single-stranded DNA. They play crucial biological functions and are implicated in neurological diseases, including ataxias, amyotrophic lateral sclerosis, nucleotide expansion disorders (Friedreich ataxia and fragile X syndrome), and cancer. Currently, it is unclear which mechanisms cause R-loop structures to become pathogenic. The RNA/DNA helicase senataxin (SETX) is one of the best characterised R-loop-binding factors in vivo. Mutations in SETX are linked to two neurodegenerative disorders: ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis type 4 (ALS4). SETX is known to play a role in transcription, neurogenesis, and antiviral response. Here, we review the causes of R-loop dysregulation in neurodegenerative diseases and how these structures contribute to pathomechanisms. We will discuss the importance of SETX as a genome guardian in suppressing aberrant R-loop formation and analyse how SETX mutations can lead to neurodegeneration in AOA2/ALS4. Finally, we will discuss the implications for other R-loop-associated neurodegenerative diseases and point to future therapeutic approaches to treat these disorders.

Increased global transcription activity as a mechanism of replication stress in cancer.

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

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.

Mechanisms of transcriptional dysregulation in repeat expansion disorders.

Approximately 40 human diseases are associated with expansion of repeat sequences. These expansions can reside within coding or non-coding parts of the genes, affecting the host gene function. The presence of such expansions results in the production of toxic RNA and/or protein or causes transcriptional repression and silencing of the host gene. Although the molecular mechanisms of expansion diseases are not well understood, mounting evidence suggests that transcription through expanded repeats plays an essential role in disease pathology. The presence of an expansion can affect RNA polymerase transcription, leading to dysregulation of transcription-associated processes, such as RNA splicing, formation of RNA/DNA hybrids (R-loops), production of antisense, short non-coding and bidirectional RNA transcripts. In the present review, we summarize current advances in this field and discuss possible roles of transcriptional defects in disease pathology.

R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome.

Friedreich ataxia (FRDA) and Fragile X syndrome (FXS) are among 40 diseases associated with expansion of repeated sequences (TREDs). Although their molecular pathology is not well understood, formation of repressive chromatin and unusual DNA structures over repeat regions were proposed to play a role. Our study now shows that RNA/DNA hybrids (R-loops) form in patient cells on expanded repeats of endogenous FXN and FMR1 genes, associated with FRDA and FXS. These transcription-dependent R-loops are stable, co-localise with repressive H3K9me2 chromatin mark and impede RNA Polymerase II transcription in patient cells. We investigated the interplay between repressive chromatin marks and R-loops on the FXN gene. We show that decrease in repressive H3K9me2 chromatin mark has no effect on R-loop levels. Importantly, increasing R-loop levels by treatment with DNA topoisomerase inhibitor camptothecin leads to up-regulation of repressive chromatin marks, resulting in FXN transcriptional silencing. This provides a direct molecular link between R-loops and the pathology of TREDs, suggesting that R-loops act as an initial trigger to promote FXN and FMR1 silencing. Thus R-loops represent a common feature of nucleotide expansion disorders and provide a new target for therapeutic interventions.