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CytoDRIP-seq in HeLa cells

ABSTRACT: R-loops are three-stranded nucleic acid structures that have important regulatory roles in cells. Deregulation of R-loop dynamics, however, can lead to DNA damage and genome instability, which results from the action of endonucleases such as XPG on R-loops. The mechanism by which this R-loop processing occurs and the impact on the cell remain unclear. Here, we show that cells with R-loops induced by deregulation of the RNA-DNA helicase Senataxin (SETX), the breast cancer gene BRCA1, or RNA splicing, accumulate cytoplasmic RNA-DNA hybrids in an XPG-dependent manner. Sequencing of cytoplasmic hybrids upon loss of SETX shows that they are derived from a subset of nuclear RNA-DNA hybrids that have longer lifetimes, signal on both sense and antisense strands and contain regions with switches in nucleotide skew. We further show that induced cytoplasmic hybrids bind the pattern recognition receptors cGAS and TLR3, activating IRF3 and inducing apoptosis. Ataxia oculomotor apraxia type 2 (AOA2) patient cells which harbor a mutated SETX also exhibit XPG-dependent activation of the innate immune response. These findings identify RNA-DNA hybrids as novel, immunogenic nucleic acid species which aberrantly accumulate in the cytoplasm of cells upon R-loop processing, and can ultimately induce cell apoptosis. We anticipate that aberrant R-loop processing and subsequent innate immune activation may be a pathological process that contributes to many disease states, such as neurodegeneration and cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178841 | GEO | 2022/10/26

REPOSITORIES: GEO

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

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2022-11-02 | PXD034331 | Pride

Epigenomic profiling of SETX and Rloops in meiotic germ cells by CUT&Tag

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

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2020-05-29 | GSE134084 | GEO

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

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2022-05-22 | GSE147886 | GEO

ChIP-Seq in mouse embryonic stem cells of endogenous HA-tagged Tet1

Project description:R-loops are prevalent three-stranded nucleic acid structures, composed of a DNA:RNA hybrid and a displaced single-stranded DNA loop. Although aberrant R-loop formation is a threat to genome stability, R-loops are enriched at CpG islands (CGI) and are implicated in maintaining CGI promoters unmethylated. Mapping of genomic regions where Tet1 binds in an R-loop dependent manner we here identify almost exclusively CGIs. The results suggest that R-loops are genomic address labels for TET1.

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Project description:SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.

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