RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics.

DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

Looping the (R) Loop in DSB Repair via RNA Methylation.

In this issue of Molecular Cell, Zhang et al. (2020) reveal that ATM triggers RNA methylation of DNA-RNA hybrids formed at double-strand breaks (DSBs) to modulate repair, adding a new layer of complexity to RNA's role in the DNA damage response.

Transcription-mediated replication hindrance: a major driver of genome instability.

Genome replication involves dealing with obstacles that can result from DNA damage but also from chromatin alterations, topological stress, tightly bound proteins or non-B DNA structures such as R loops. Experimental evidence reveals that an engaged transcription machinery at the DNA can either enhance such obstacles or be an obstacle itself. Thus, transcription can become a potentially hazardous process promoting localized replication fork hindrance and stress, which would ultimately cause genome instability, a hallmark of cancer cells. Understanding the causes behind transcription-replication conflicts as well as how the cell resolves them to sustain genome integrity is the aim of this review.

The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores.

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Break-induced RNA-DNA hybrids (BIRDHs) in homologous recombination: friend or foe?

Double-strand breaks (DSBs) are the most harmful DNA lesions, with a strong impact on cell proliferation and genome integrity. Depending on cell cycle stage, DSBs are preferentially repaired by non-homologous end joining or homologous recombination (HR). In recent years, numerous reports have revealed that DSBs enhance DNA-RNA hybrid formation around the break site. We call these hybrids "break-induced RNA-DNA hybrids" (BIRDHs) to differentiate them from sporadic R-loops consisting of DNA-RNA hybrids and a displaced single-strand DNA occurring co-transcriptionally in intact DNA. Here, we review and discuss the most relevant data about BIRDHs, with a focus on two main questions raised: (i) whether BIRDHs form by de novo transcription after a DSB or by a pre-existing nascent RNA in DNA regions undergoing transcription and (ii) whether they have a positive role in HR or are just obstacles to HR accidentally generated as an intrinsic risk of transcription. We aim to provide a comprehensive view of the exciting and yet unresolved questions about the source and impact of BIRDHs in the cell.

Histone Mutants Separate R Loop Formation from Genome Instability Induction.

R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.

VID22 counteracts G-quadruplex-induced genome instability.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.

Expression of immune checkpoint molecules programmed death protein 1, programmed death-ligand 1 and inducible T-cell co-stimulator in mycosis fungoides and Sézary syndrome: association with disease stage and clinical outcome.

BACKGROUND: The relationship between immune checkpoint status and disease outcome is a major focus of research in cutaneous T-cell lymphoma (CTCL), a disfiguring neoplastic dermatological disorder. Mycosis fungoides (MF) and Sézary syndrome (SS) are the two most common types of CTCL. OBJECTIVES: The aim was to evaluate the immune checkpoint markers programmed death protein 1 (PD1), inducible T-cell co-stimulator (ICOS) and programmed death-ligand 1 (PD-L1) in skin biopsies from patients with CTCL relative to disease stage and overall survival. METHODS: This consecutive case series enrolled 47 patients: 57% had stage IA-IIA disease and 43% had stage IIB-IVA2 disease (including seven with SS). RESULTS: PD1, PD-L1 and ICOS expression was seen in all biopsies. Notably, PD-L1 was predominantly expressed on histiocytes/macrophages, but focal expression on CTCL cells was seen. High expression of either ICOS or PD-L1 was associated with advanced-stage disease (P = 0·007 for both) and with the appearance of large-cell transformation (LCT), a histopathological feature associated with a poor prognosis (ICOS: P = 0·02; PD-L1: P = 0·002). PD1 expression was not significantly associated with disease stage (P = 0·12) or LCT (P = 0·49), but expression was high in SS biopsies. A high combined checkpoint marker score (PD1, PD-L1 and ICOS) was associated with advanced-stage disease (P = 0·001), LCT (P = 0·021) and lower overall survival (P = 0·014). CONCLUSIONS: These findings demonstrate the existence of a complex immunoregulatory microenvironment in CTCL and support the development of immunotherapies targeting ICOS and PD-L1 in advanced disease.

Real-Time Genomic Surveillance for SARS-CoV-2 Variants of Concern, Uruguay.

We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.

Origin matters: spontaneous DNA-RNA hybrids do not form in trans as a source of genome instability.

Multiple exogenous and endogenous genotoxic agents threaten the integrity of the genome, but one major source of spontaneous DNA damage is the formation of unscheduled DNA-RNA hybrids. These can be genetically detected by their ability to induce recombination. The origin of spontaneous hybrids has been mainly attributed to the nascent RNA formed co-transcriptionally in cis invading its own DNA template. However, it was unclear whether hybrids could also be spontaneously generated by RNA produced in a different locus (in trans). Using new genetic systems in the yeast Saccharomyces cerevisiae, we recently tested whether hybrids could be formed in trans and compromise genome integrity. Whereas we detected recombinogenic DNA-RNA hybrids in cis and in a Rad51-independent manner, we found no evidence for recombinogenic DNA-RNA hybrids to be formed with RNAs produced in trans. Here, we further discuss the implications in the field for the origin of genetic instability and the threats coming from RNAs.

The DNA damage response acts as a safeguard against harmful DNA-RNA hybrids of different origins.

Despite playing physiological roles in specific situations, DNA-RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA-RNA hybrids, here we screen an siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs causing DNA-RNA hybrid accumulation and a significant increase in hybrid-dependent DNA breakage. We identify post-replicative repair and DNA damage checkpoint factors, including those of the ATM/CHK2 and ATR/CHK1 pathways. Thus, spontaneous DNA-RNA hybrids are likely a major source of replication stress, but they can also accumulate and menace genome integrity as a consequence of unrepaired DSBs and post-replicative ssDNA gaps in normal cells. We show that DNA-RNA hybrid accumulation correlates with increased DNA damage and chromatin compaction marks. Our results suggest that different mechanisms can lead to DNA-RNA hybrids with distinct consequences for replication and DNA dynamics at each cell cycle stage and support the conclusion that DNA-RNA hybrids are a common source of spontaneous DNA damage that remains unsolved under a deficient DDR.

Skin adnexal carcinoma with BRD3-NUTM2B fusion.

NUT carcinomas are genetically defined epithelial neoplasms. Most tumors harbor fusions of NUTM1 with BRD4 or BRD3. Their histopathologic features have been predominantly reported as undifferentiated or poorly differentiated squamous cell carcinoma, and clinically they tend to be aggressive cancers. However, recent studies have revealed a broader spectrum of NUTM1-rearranged neoplasms with several new fusion partners and associated variable histopathologic phenotypes and clinical behaviors, including benign and malignant cutaneous poroid tumors. We report herein a primary invasive carcinoma of skin adnexal origin with a previously undescribed fusion between BRD3 and NUTM2B. The tumor occurred on the shoulder of a 7-year-old girl and was excised with negative margins. A sentinel lymph node was positive. After follow-up of 23 months, and without systemic treatment, the child remains free of tumor. This case expands the spectrum of NUT carcinomas by including a skin adnexal variant with follicular infundibular differentiation, a novel genomic aberration, and preliminary evidence of a less aggressive clinical course.

Folliculotropic mycosis fungoides driven by DOCK8 immunodeficiency syndrome.

DOCK8 immunodeficiency syndrome (DIDS) represents a rare primary immunodeficiency associated with cutaneous viral infections, allergy, and increased risk of malignancy. We report a case of folliculotropic mycosis fungoides with spontaneous resolution occurring in a patient with DIDS.

A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination.

Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage.

RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.

Relief of intractable pruritus with romidepsin in patients with cutaneous T-cell lymphoma: A series of four cases.

Cutaneous T-cell lymphomas (CTCL) are a relatively rare and heterogeneous group of non-Hodgkin lymphomas that typically present in the skin. The majority of patients with CTCL experience pruritus, which can interfere with daily activities, significantly impact quality of life, and is typically uncontrolled by standard anti-itch therapies. Several lymphoma treatments have reported anti-pruritic effects including romidepsin, a potent class 1 selective histone deacetylase inhibitor approved for the treatment of patients with CTCL who have had at least one prior systemic therapy. Here, we describe the cases of four patients with debilitating and refractory pruritus that were resolved with romidepsin. Resolution of pruritus was observed in both clinical responders and nonresponders, and dose modification was used successfully to manage adverse events and for maintenance treatment. The potential for pruritus relief with romidepsin should be considered when treating patients with CTCL.

Yeast Sen1 helicase protects the genome from transcription-associated instability.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.

Progression of undiagnosed cutaneous lymphoma after anti-tumor necrosis factor-alpha therapy.

BACKGROUND: Cutaneous lymphoma diagnosed after anti-tumor necrosis factor-α therapy (anti-TNF-α) has been reported in the literature, yet a clear link between both events remains elusive. OBJECTIVE: To review our experience with cutaneous lymphoma diagnosed during or after the use of anti-TNF-α therapies. METHODS: This is a multicenter retrospective study and a literature review. RESULTS: A total of 22 cases, including 20 cutaneous T-cell lymphomas (CTCLs) and 2 cutaneous B-cell lymphomas, were identified. In the CTCL group, 75% of the patients received an anti-TNF-α agent for a presumed inflammatory skin condition. Mycosis fungoides and Sézary syndrome were the most common subtypes of CTCL diagnosed. Advanced disease (stage IIB to IVA) was commonly seen at time of diagnosis and required aggressive therapy, including stem cell transplant in 3 patients; 2 patients in whom cutaneous B-cell lymphomas was diagnosed had an indolent course. A total of 31 cases were gathered from a literature search. LIMITATIONS: This is a retrospective study. CONCLUSIONS: Our findings suggest that the disease of most of the identified patients was misdiagnosed as psoriasis or eczema; therefore, a comprehensive morphologic and molecular review of skin biopsy specimens and peripheral blood samples should be considered before initiation of anti-TNF-α therapy in patients with poorly defined dermatitis or atypical presentations of psoriasis.