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.
Highlights
Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks
We propose that the accumulation of co-transcriptional R-loops, and replication stress, double-strand breaks (DSBs), and an inflammatory response may collectively contribute to the development of familial acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with mutated DDX41
The advantages of RNA–DNA Proximity Proteomics (RDProx) are manifold: (1) labeling of R-loop-proximal proteins is performed in vivo, which ensures that R-loops, chromatin, and cellular compartments remain intact; (2) chromatin-associated proteins that are difficult to solubilize are amenable to the analysis, and (3) low affinity and transient interactions are detected
Summary
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 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. 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. Co-transcriptional R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. In addition to the regulatory functions of R-loops in transcription, DNA repair, telomere maintenance, and chromosome segregation, these non-B DNA structures can be drivers of genomic instability[17,18,19,20,21,22]. R-loops can be formed as a consequence of head-on transcription–replication conflicts[25,26,27]
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