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

Abstract

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct 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 RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

Integrative analysis and prediction of human R-loop binding proteins

“…1a; Supplementary Table 1). Similarly, to develop a consensus proximity-based RLBP dataset, we intersected the Mosler et al (2021) dataset (660 proteins) and the Yan et al (2022) dataset (440 proteins) to reveal 101 proteins that have high R-loop binding affinity identified via Prox-MS approaches (Prox-MS RLBPs). Hereon, the terms RLBPs refers to both IP-MS RLBPs and Prox-MS RLBPs unless specified otherwise.…”

“…Datasets for curating the IP-MS RLBPs list were taken from Cristini et al (2018) , the R-loop attracted proteins dataset from Wang et al (2018) and Wu et al (2021 ) while the Prox-MS RLBPs list were taken from Mosler et al (2021) and Yan et al (2022 ). Protein domain information was extracted from Pfam version 32.0 using an E value <0.001 ( El-Gebali et al 2019 ).…”

“…Overall, these 3 studies that rely on immunoprecipitation coupled with mass spectrometry (IP-MS) identified hundreds of candidate’s RLBPs and overlapped by >200 proteins creating a higher confidence set of proteins that may preferentially recognize R-loops. In parallel, 2 studies applied a proximity-labeling approach to characterize the R-loop interactome where biotinylated proteins were purified using streptavidin affinity coupled with mass spectrometry (Prox-MS) ( Mosler et al 2021 ; Yan et al 2022 ). These Prox-MS studies revealed a new overlapping set of R-loop interactors (>100 proteins) indicating that while IP-MS techniques have their advantages, they enrich for stable R-loop-protein interactions and might lose transient interactions which Prox-MS can capture.…”

Secondary structures in RNA synthesis, splicing and translation

“… DDX2A plays an essential role in spermatogenesis, whereas DDX2B is essential for mouse viability [123] . DDX41 R-loops Resolves R-loops that emerge during transcription [124] . R-loop accumulation and genomic instability due to knock down of DDX41 [124] .…”

“… DDX41 R-loops Resolves R-loops that emerge during transcription [124] . R-loop accumulation and genomic instability due to knock down of DDX41 [124] . DDX39B ( UAP56 ) R-loops Spliceosomal helicase with roles in the removal of R-loops [125] .…”