cytoDRIP-seq in HeLa cells

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. Overall design: IgG and S9.6 IP cytoplasmic extracts following control and SETX knockdown in HeLa cells