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

Abstract

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) and permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify 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 inflammatory signaling. DDX41 opposes the accumulation of R-loops by unwinding RNA-DNA hybrids at gene promoters and its loss leads to upregulation 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 R-loops at CpG island promoters, altered TGFβ and NOTCH signaling, and inflammatory response contribute to the development of familial AML with mutated DDX41.

DEAD-Box RNA Helicases and Genome Stability

“…Detection of R-loops via immunofluorescence demonstrated that R-loops levels were increased in ddx41 loss-of-function mutants in zebrafish embryos, which could be reversed upon RNaseHI expression [63]. Similar to findings in zebrafish, DDX41 was found to suppress R-loop accumulation in human HEK-293 cells, suggesting a possible conservation of mechanism [67]. Resolving R-loops appears important for the genomic stability function of DDX41 as overexpression of RNaseH1 in these cells partially rescued the accumulation of DNA breaks upon DDX41 knockdown.…”

“…DDX41 has multiple cellular functions associated with innate immune signaling, inflammation, R-loop metabolism and splicing [62][63][64][65][66]. Disrupting DDX41 in human and zebrafish models is associated with the accumulation of DNA breaks [63,67]. In zebrafish, ddx41 loss-of-function mutants (ddx41 sa14887 ) develop anemia due to defective erythropoiesis.…”

“…Interestingly, DDX41 was also identified in an R-loop interactome, suggesting a role in the regulation of DNA:RNA hybrids [65,67]. Detection of R-loops via immunofluorescence demonstrated that R-loops levels were increased in ddx41 loss-of-function mutants in zebrafish embryos, which could be reversed upon RNaseHI expression [63].…”

Histone H3 deacetylation promotes host cell viability for efficient infection by Listeria monocytogenes

“…Transcriptional repression is also a feature of the DDR as transcriptional machinery can interfere with DNA repair. Interestingly, in silico analysis shows that many SIRT2 regulated sequences contain or are predicted to contain R-loops; additionally there are multiple studies which demonstrate that TDP-43 localises to and interacts with R-loops [53][54][55]. We also show that TDP-43 is recruited to the same loci as SIRT2, however, as we observe global cellular effects such as H3K18 deacetylation by immunoblot and changes to cell viability, we predict that other loci in addition to the subset of TSSs we previously identified are also likely to be targeted.…”

Roles of RNA-binding proteins in neurological disorders, COVID-19, and cancer

“…In embryonic stem cells, RBPs represent half of the chromatin proteome, cocompartmentalize with RNA polymerase (Pol) II at promoters and enhancer, and connect RNA to the transcription machinery [38]. The RBP DDX41 is a tumor suppressor that opposes double-strand DNA breaks, genomic instability and R-loop-dependent replication stress by preferentially binding RNA-DNA hybrids and unwinding RNA-DNA hybrids in R-loops, and also reduces fragility of DNA in promoter regions [39]. RBP HNRNPC is demonstrated to interact with circXRCC5 to encourage circXRCC5 biogenesis that was proved to foster Gastric cancer progression [40].…”