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The extruded non-template strand determines the architecture of R-loops

ABSTRACT: We show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

ORGANISM(S): Escherichia coli

PROVIDER: GSE129936 | GEO | 2019/04/23

REPOSITORIES: GEO

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The extruded non-template strand determines the architecture of R-loops

Project description:The extruded non-template strand determines the architecture of R-loops

| PRJNA533230 | ENA

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

Two Distinct Types of RNA:DNA Hybrids Revealed by Evaluating Divergent “R-loop” Profiles Detected with Different Mapping Strategies

Project description:R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

2022-05-22 | GSE147886 | GEO

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

Proteomic analysis of DNA synthesis on a structured DNA template in human cellular extracts: Interplay between NHEJ and replication-associated proteins

Project description:It has been established that short inverted repeats (SIRs) trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with this structured DNA is unknown. We have previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded DNA template is transiently blocked at DNA hairpins [1](Schmutz et al., 2007). Here, we report the proteomic analysis of proteins bound to the DNA template providing evidence that proteins of the NHEJ pathway, particularly the DNA-PK complex (composed of DNA-PKcs and the Ku70/Ku80 dimer) recognize structured single-stranded DNA. DNA-PKcs inhibition results in the mobilization on the template DNA of the DNA-PK complex, along with other proteins acting downstream in the NHEJ pathway, especially the XRCC4-DNA Ligase 4 complex and the recently identified cofactor PAXX. The retention of NHEJ factors to the template DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that NHEJ proteins may hinder the progression of the DNA synthesis at these sites. Conversely, hijacking of the DNA-PK complex by double-stranded oligos (dsO) results in a large removal of the pausing sites and an elevated DNA extension efficiency. Overall these results raise the possibility that, upon binding to DNA hairpins formed onto ssDNA during fork progression, the DNA-PK complex may play some role in replication fork dynamics in vivo, role that is however not related to repair of double-strand breaks.

2020-02-04 | PXD011156 | Pride

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

Project description:R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase H-based approach, revealing predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end, and by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

2017-10-12 | GSE97072 | GEO

Characterization of R-loop-interacting proteins in embryonic stem cells reveals roles in ribosomal RNA processing and gene expression

Project description:Chromatin-associated RNAs have diverse roles in the nucleus. However, their mechanisms of action are poorly understood, in part due to the inability to identify proteins that specifically associate with chromatin-bound RNAs. Here, we address this problem for a subset of chromatin-associated RNAs that form R-loops—RNA-DNA hybrid structures that include a displaced strand of single-stranded DNA. R-loops generally form co-transcriptionally and have important roles in regulation of gene expression, immunoglobulin class switching, and other processes. However, unresolved R-loops can lead to DNA damage and chromosome instability. To identify factors that may bind and potentially regulate R-loop accumulation or mediate R-loop-dependent functions, we used a comparative immunoprecipitation/mass spectrometry approach, with and without RNA-protein crosslinking, to identify a stringent set of R-loop-binding proteins in mouse embryonic stem cells. We identified 365 R-loop-interacting proteins, which were highly enriched for proteins with predicted RNA-binding functions. We characterized several R-loop-interacting proteins of the DEAD-box family of RNA helicases and found that these proteins localize to the nucleolus and, to a lesser degree, the nucleus. Consistent with their localization patterns, we found that these helicases are required for ribosomal RNA processing and regulation of gene expression. Surprisingly, depletion of these helicases resulted in misregulation of highly overlapping sets of protein-coding genes, including many genes that function in differentiation and development. We conclude that R-loop-interacting DEAD-box helicases have non-redundant roles that are critical for maintaining the normal embryonic stem cell transcriptome.

2021-09-17 | PXD022697 | Pride

Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

Project description:R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.

2017-10-25 | GSE98886 | GEO

Splicing Inhibition Reveals Novel Roles for SF3B1 in Transcription Dynamics and R-Loop Metabolism

Project description:Efficient co-transcriptional splicing has been proposed to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. To test this, we inhibited the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells and mapped R-loop genomic distributions. PladB caused widespread intron retention, as expected, and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these two events, arguing that unspliced introns do not cause excessive R-loops. Instead, R-loop gains were driven by a loss of transcription termination over a specific subset of stress-response genes, defining a new class of “downstream of genes” (DoG) aberrant R-loops. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss over thousands of genes, resulting from a profound loss of transcription elongation. Thus, acute splicing inhibition triggered profound and contrasting alterations in transcriptional dynamics, which were reflected in the global R-loop landscape.

2020-06-10 | GSE148768 | GEO

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2019-11-11 | GSE138200 | GEO

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