Cytoplasmic R-loop Processing–Derived RNA–DNA Hybrids Are Immunogenic

R-loops, RNA–DNA hybrid–containing nucleic acid structures, have important cellular functions, but unintended R-loop formation can block effective DNA replication and transcription as well as lead to double-stranded breaks, genome instability, senescence, and cell death through the action of endonucleases. However, the mechanisms and cellular consequences of the endocytic processing of R-loops remain incompletely understood. Crossley, Song, and colleagues sought to further study R-loop processing and revealed a new RNA–DNA hybrid population in the cytoplasm that was identified as a product of R-loop processing. Accumulation of these cytoplasmic hybrid fragments was dependent on XPG and XPF endonucleases, while depletion of factors that suppress R-loop levels, including SETX and BRCA1, also increased cytoplasmic accumulation of these fragments. Sequencing of this RNA–DNA hybrid population revealed that they are generated from a small subset of nuclear R-loops, with only a small portion of these R-loops being susceptible to XPG-dependent processing in the absence of SETX. Moreover, evaluation of their stability after SETX loss indicated their long half-lives. As previous studies have demonstrated that R-loop deregulation is linked to immune responses through pattern recognition receptors, investigation into the role that these cytoplasmic hybrids play in this process revealed that IRF3 immune signaling is triggered after SETX or BRCA1 depletion. Additionally, induction of cleaved PARP and caspase 3 activity was observed after loss of SETX or BRCA1, which could be blocked by XPG or XPF loss. The proinflammatory and apoptosis factor TNF was also induced, which further supports that these cytoplasmic hybrids contribute to both innate immune activation and apoptosis. Mechanistically, cGAS and TLR3 were both found to be able to recognize endogenous, R-loop–derived cytoplasmic RNA–DNA hybrids, which subsequently leads to IRF3 activation and apoptosis induction. Finally, accumulation of these R-loop–derived cytoplasmic RNA–DNA hybrids was confirmed in models of human disease, including a model of ovarian cancer. In summary, the results of this study show that RNA–DNA hybrids generated from R-loop processing are immunogenic and are relevant to human disease, including cancer.

Crossley MP, Song C, Bocek MJ, Choi JH, Kousorous J, Sathirachinda A, et al. R-loop-derived cytoplasmic RNA–DNA hybrids activate an immune response. Nature 2023;613:187–94.

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