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

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 HRAS(V12) 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 HRAS(V12), 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

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

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

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. 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

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

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

RNA UK 2012 An Independent Meeting held at The Burnside Hotel

Abstract Most human genes transcribed by RNA Pol II (polymerase II) contain short exons separated by long tracts of non-coding intronic sequences. In addition to their role in generating proteomic diversity through the process of alternative splicing, intronic sequences host many ncRNAs (non-coding RNAs), involved in various gene regulation processes. miRNAs (microRNAs) are short ncRNAs that mediate either mRNA transcript translational repression and/or degradation. Between 50 and 80 % of miRNAs are encoded within introns of host mRNA genes. This observation suggests that there is co-regulation between the miRNA biogenesis and pre-mRNA splicing processes. The present review summarizes current advances in this field and discusses possible roles for intronic co-transcriptional cleavage events in the regulation of human gene expression. Intronic miRNA (microRNA) biogenesis miRNAs are short non-coding regulatory molecules involved in diverse biological processes in humans. In the human genome, miRNA sequences can be present either as part of independent Pol II (polymerase II) transcription units or within annotated &apos;host&apos; genes. Of the 50-80 % of human miRNAs that are found in introns, most are preferentially located near the middle of the intron During miRNA synthesis, Drosha, the main nuclear RNase III-like enzyme in humans, together with its cofactor DGCR8 (DiGeorge syndrome critical region gene 8)/Pasha, functions as a part of the microprocessor complex that generates a 70 nt pre-miRNA precursor molecule Key words: co-transcriptional processing, Drosha, intronic microRNA (intronic miRNA), splicing. Abbreviations used: DGCR8, DiGeorge syndrome critical region gene 8; miRNA, microRNA; Pol II, polymerase II. 1 email [email protected] miRNA biogenesis is a regulated process and a number of RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) A1, Lin28, Smad proteins and KSRP (KH-type splicing regulatory protein) have been shown to positively or negatively regulate miRNA production (reviewed in Co-transcriptional processing of intronic miRNAs Studies have shown that miRNA biogenesis in the nucleus occurs co-transcriptionally from both independently transcribed and intron-encoded primary miRNA

R-loops and human diseases.

<p>The diagram depicts the role of R-loops in human diseases. Loss of wild type protein function is depicted by red crosses. <b>A.</b> Ataxia and motor neuron diseases. Mutations in human RNA/DNA helicase senataxin are associated with AOA2/ALS4 disorders and lead to R-loop accumulation and defects in transcriptional termination by Pol II <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-SkourtiStathaki2" target="_blank">[16]</a>, the maintenance of genome integrity <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Yuce1" target="_blank">[46]</a>, meiotic recombination during spermatogenesis, gene silencing during meiotic sex chromosome inactivation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Becherel1" target="_blank">[14]</a>, and neuronal differentiation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Vantaggiato1" target="_blank">[49]</a>. <b>B.</b> Aicardi-Goutières syndrome (AGS). AGS is associated with mutations in all three subunits of RNase H2, ssDNA 3′–5′ exonuclease TREX1 (DNASEIII), dsRNA-editing enzyme ADAR1, and dNTP triphosphatase SAMHD1; these trigger accumulation of unprocessed nucleic acids, including genomic DNA with incorporated ribonucleotides, R-loops, and retroelement-derived nucleic acids, and result in the immune response characteristic of AGS <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Rabe1" target="_blank">[65]</a>. <b>C.</b> Trinucleotide expansion diseases. R-loops form over expanded repeats and result in decreased initiation and elongation of RNA Pol II and formation of repressive chromatin marks, which silence the host gene containing expanded repeats <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Groh1" target="_blank">[75]</a>. <b>D.</b> Genome instability in cancer. Loss of proteins protecting against abnormal R-loop accumulation, such as FIP1L1, leads to genome instability, one hallmark of cancer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Stirling1" target="_blank">[31]</a>. Yellow stars denote double-stranded DNA breaks. <b>E.</b> AID-mediated mutagenesis and translocations in cancer. Single-stranded DNA in R-loops is a substrate for cytidine deamination by activation-induced cytidine deaminase, leading to mutagenesis as indicated by orange stars <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Yu1" target="_blank">[21]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Ruiz1" target="_blank">[88]</a>. These mutations can cause DSB formation, leading to chromosomal translocations. The <i>IgH/c-MYC</i> translocation brings the strong <i>IgH</i> enhancers, shown as yellow box, close to <i>c-MYC</i>, leading to its overexpression in Burkitt's lymphoma <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Robbiani1" target="_blank">[87]</a>. Transcription of <i>IgH/c-MYC</i> starts from a previously inactive promoter downstream of the translocation break point. The <i>IgH</i> locus is depicted in blue, <i>c-MYC</i> gene is in grey. The translocation breakpoint is indicated by a dashed black line. <b>F.</b> Senescence. R-loops formed by the noncoding RNA TERRA accumulate at telomeres in cells deficient of Hpr1 and RNase H. In the absence of telomerase, these R-loops promote Rad52-dependent telomere elongation and delayed senescence. In the absence of telomerase and Rad52, R-loops promote telomere shortening and premature senescence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Balk1" target="_blank">[94]</a>.</p

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types

Alternative splicing of α-tropomyosin (α-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitutions of the UGC-containing upstream regulatory element (URE) with other UGC elements disrupted the α-TM splicing pattern in transfected cells. Multimerisation of the URE caused enhanced exon skipping in SM and various non-SM cells. In the presence of multiple UREs the degree of splicing regulation was decreased due to the high levels of exon skipping in non-SM cell lines. These results suggest that the URE is not an intrinsically SM- specific element, but that its functional strength is fine tuned to exploit differences in the activities of regulatory factors between SM and other cell types. Co-transfection of tropomyosin reporters with members of the CUG-binding protein family, which are candidate URE-binding proteins, indicated that these factors do not mediate repression of tropomyosin exon 3

History of R-loop research.

<p>The diagram depicts major developments in the R-loop field and diseases associated with R-loop dysregulation.</p