R-loop-derived cytoplasmic RNA–DNA hybrids activate an immune response

R-loops are RNA–DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability 1 , which has been linked to the action of endonucleases such as XPG 2 – 4 . However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA–DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA–DNA helicase senataxin ( SETX ) or the breast cancer gene BRCA1 (refs. 5 – 7 ), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8 ), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX -mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9 ) and in BRCA1 -mutated cancer cells 10 . These findings establish RNA–DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.