R-loops

The ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds to and immobilises the RAE1 protein on the cytoplasmic membranes, thereby blocking mRNA transport from the nucleus to the cytoplasm. In all these cases, the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands...

Drugs targeting the DNA damage response (DDR) are widely used in cancer therapy, but resistance to these drugs remains a major clinical challenge. Here, we show that SYCP2, a meiotic protein in the synaptonemal complex, is aberrantly and commonly expressed in breast and ovarian cancers and associated with broad resistance to DDR drugs. Mechanistically, SYCP2 enhances the repair of DNA double-strand breaks (DSBs) through transcription-coupled homologous recombination (TC-HR). SYCP2 promotes R-loop formation at DSBs and facilitates RAD51 recruitment independently of BRCA1...

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employ several techniques to map DSBs, R-loops, and topoisomerase 1 cleavage complex (TOP1cc) to comprehensively investigate the interplay between transcription, DSBs, topoisomerase 1 (TOP1), and R-loops. Our findings reveal the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops. Remarkably, transcription-associated DSBs at these loci are significantly reduced upon depletion of R-loops and TOP1, uncovering the pivotal roles of TOP1 and R-loops in transcriptional DSB formation...

Autosomal dominant polycystic kidney disease results from the loss of the PKD1 gene product, polycystin 1. Regulatory mechanisms are unresolved, but an apparent G/C sequence bias in the gene is consistent with co-transcriptional R-loop formation. R-loops regulate gene expression and stability, and they form when newly synthesized RNA extensively pairs with the template DNA to displace the non-template strand. In this study, we tested two human PKD1 sequences for co-transcriptional R-loop formation in vitro...

R-loops are three-stranded structures that can pose threats to genome stability. RNase H1 precisely recognizes R-loops to drive their resolution within the genome, but the underlying mechanism is unclear. Here, we report that ARID1A recognizes R-loops with high affinity in an ATM-dependent manner. ARID1A recruits METTL3 and METTL14 to the R-loop, leading to the m6A methylation of R-loop RNA. This m6A modification facilitates the recruitment of RNase H1 to the R-loop, driving its resolution and promoting DNA end resection at DSBs, thereby ensuring genome stability...

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated-motifs (iMs), and R-loops (RLs). In this paper, we utilized computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts...

DDX41 is a DEAD-box helicase and is conserved across species. Mutations in DDX41 have been associated with myeloid neoplasms, including myelodysplastic syndrome and acute myeloid leukemia. Though its pathogenesis is not completely known, DDX41 has been shown to have many cellular roles, including in pre-mRNA splicing, innate immune sensing, ribosome biogenesis, translational regulation, and R-loop metabolism. In this review, we will summarize the latest understandings regarding the various roles of DDX41, as well as highlight challenges associated with drug development to target DDX41...

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures...

R-loops (RNA-DNA hybrids with displaced single-stranded DNA) have emerged as a potent source of DNA damage and genomic instability. The termination of defective RNA polymerase II (RNAPII) is one of the major sources of R-loop formation. 5'-3'-exoribonuclease 2 (XRN2) promotes genome-wide efficient RNAPII termination, and XRN2-deficient cells exhibit increased DNA damage emanating from elevated R-loops. Recently, we showed that DNA damage instigated by XRN2 depletion in human fibroblast cells resulted in enhanced poly(ADP-ribose) polymerase 1 (PARP1) activity...

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2Δ/Y ) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions...

Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells...

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis...

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops...

Dot-blot analysis is a technique that allows for fast and convenient detection and identification of nucleic acids and proteins. Here, we provide a guide for nucleic acid isolation from eukaryotic cells and sample processing to detect RNA/DNA hybrids. We then provide detailed steps to quantify dot signal intensity. This protocol can be adapted for screening conditions that result in the accumulation of R-loops. For complete details on the use and execution of this protocol, please refer to Smith et al...

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast...

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays important roles in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive...

Loss of BRCA2 (BReast CAncer 2) is lethal for normal cells. Yet, it remains poorly understood how in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene, MLH1 as a genetic interactor of BRCA2 whose over-expression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments...

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes...

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The Repeat Expansion Diseases (REDs) arise from expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion and whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase ( GLS ) gene...