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UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

ABSTRACT: Non-scheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R loop accumulation. One mechanism relies on the conserved THO complex in association with co-transcriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R loop removal. We show that UAP56 depletion causes R loop accumulation, R loop-mediated genome instability and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and that its overexpression suppresses R loops and genome instability induced by depleting 5 different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful co-transcriptional RNA structures that otherwise would block transcription and replication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE127979 | GEO | 2020/05/25

REPOSITORIES: GEO

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UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

Project description:UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

| PRJNA525976 | ENA

DDX17 helicase promotes resolution of R-loop-mediated transcription-replication conflicts in human cells

Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.

2022-11-02 | PXD034331 | Pride

ATAD5 regulates R-loop by maintaining RNA helicase level and unloading PCNA at the replication fork

Project description:R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflicts (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

2021-09-09 | PXD018207 | Pride

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instabilityR-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Project description:Transcription can pose a threat to genomic stability through the formation of R-loops that obstruct the progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RDProx to identify proteins that regulate R-loops in human cells. RDProx relies on the expression of the hybrid-binding domain (HBD) of Ribonuclease H1 (RNaseH1) fused to the ascorbate peroxidase (APEX2), which permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We associated R-loop regulation with different cellular proteins and identified a role of the tumor suppressor DEAD box protein 41 (DDX41) in opposing R-loop-dependent genomic instability. Depletion of DDX41 resulted in replication stress, double strand breaks and increased inflammatory signaling. Furthermore, DDX41 opposes accumulation of R-loops at gene promoters and its loss leads to upregulated expression of TGFβ and NOTCH signaling genes. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia (AML) in adulthood. We propose that accumulation of co-transcriptional R-loops, associated gene expression changes and inflammatory response contribute to the development of familial AML with mutated DDX41.

2021-11-25 | PXD024517 | Pride

Yra1-bound R loops cause orientation-independent transcription-replication collisions and telomere instability

Project description:R loops are an important source of genome instability largely due to its negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess cause lethality and genome instability. Here, we show that Yra1 binds RNA-DNA hybrids in vitro and when artificially overexpressed is recruited to chromatin in an RNA-DNA hybrid-dependent manner stabilizing R loops and converting them into replication obstacles in vivo. Importantly, excess of Yra1 increases R loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are co-directional or head-on. It also induces telomere shortening and senescence, consistent with a defect in telomere replication. Our results indicate that R loops form transiently in cells regardless of replication and, after stabilization by excess Yra1, they compromise genome integrity, in agreement with a two-step model of R loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.

2018-06-27 | GSE113580 | GEO

ZFP281-BRCA2 prevents R-loop accumulation during DNA replication

Project description:R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.

2022-05-31 | GSE127262 | GEO

Proximity labeling identifies a repertoire of site-specific R-loop modulators

Project description:R-loops are three stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and that contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains at R-loops. One of the most consistently enriched proteins at R-loops was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP is necessary to suppress R-loops in vivo at its genomic targets and that it resolves R-loops in vitro. Furthermore, deletion of the homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets and compromises neuronal differentiation. Notably, patient derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

2021-06-03 | MSV000087568 | MassIVE

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [tiling array]

Project description:The transposon silencing piRNAs are produced from precursors that are encoded by heterochromatic clusters and processed in the perinuclear nuage. We show that the Drosophila nuclear DEAD box protein UAP56, previously implicated in mRNA splicing and nuclear export, co-localizes with the cluster-associated HP1 homologue Rhino. Prominent nuclear foci containing Rhi and UAP56 localize directly across the nuclear envelope from Vasa, a conserved DEAD box protein and core nuage component that is required for piRNA production, and piRNA precursors immunoprecipitate with both UAP56 and Vasa. A uap56 point mutation that prevents UAP56 protein co-localization with Rhino also disrupts nuage organization, transposon silencing, and expression of dual strand piRNA clusters. By contrast, this allele significantly increases ectopic piRNAs from protein coding genes. We therefore propose that UAP56 and Vasa organize a piRNA-processing compartment that spans the nuclear envelope, increasing the efficiency and specificity of piRNA biogenesis. 3 replicates of each sample (uap56, vasa), total RNA samples hybridized to tiling array.

2012-11-28 | E-GEOD-35636 | biostudies-arrayexpress

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [tiling array]

2012-11-28 | GSE35636 | GEO

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Project description:Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

2023-09-14 | GSE202723 | GEO

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