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Aberrant R-loops in Innate Immunity: Friend and Foe?

By Stuart P. Atkinson, Ph.D.

May 9, 2023

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Introduction: R-Loops…Friend and Foe?

Gene transcription in mammalian cells can induce the formation of “R-loops” composed of single-stranded nascent RNA molecules interacting with double-stranded DNA, which alters its typical configuration. While R-loops do play critical roles in transcriptional regulation and DNA repair (Santos-Pereira and Aguilera, 2015), their presence often associates with high levels of DNA double-stranded breaks, which can induce genome instability, senescence, and apoptosis (Crossley et al., Santos-Pereira and Aguilera, and He et al.). Known R-loop suppression mechanisms in human cells involve the unwinding of the RNA–DNA hybrid by senataxin (a helicase encoded by the SETX gene) (Skourti-Stathaki et al.) and the activity of the breast cancer type 1 susceptibility protein (encoded by the BRCA1 gene) (Hatchi et al.).

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R-loop Processing: What Becomes of the Byproducts?

Endonucleolytic processing of R-loops by endonucleases induces DNA breaks (Sollier et al., Makharashvili et al., and Cristini et al.), which leads to a general increase in DNA damage levels and genome instability (Crossley et al.); however, the fate and influence of the processed nucleic acids have remained unclear. In a recent Nature study, researchers led by Karlene A. Cimprich (Stanford University) now provide evidence that the products of R-loop-processing (by endonuclease activity) accumulate in the cytoplasm where they provoke an innate immune response that prompts cell death. Overall, this exciting research suggests that aberrant R-loop processing and subsequent innate immune activation may represent a novel disease-associated mechanism relevant to conditions such as neurodegeneration and cancer.

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The Cellular Response to R-loop Processing

Crossley and Colleagues began their study by depleting senataxin or BRCA1 in human cells to induce a high number of R-loops, which supported a significant increase in the levels of cytoplasmic RNA–DNA hybrids as the products of R-loop processing by endonuclease activity. Next-generation sequencing-based analysis of biochemically purified cytoplasmic hybrids from syntaxin-deficient cells revealed that cytoplasmic RNA–DNA hybrids originated from highly stable genomic R-loops that exhibit distinct sequence properties, consistent with convergent transcription and hybrid formation.

The study next discovered that cytoplasmic RNA–DNA hybrids bound to the cyclic GMP-AMP synthase (cGAS, cGAMP synthase) and Toll-like receptor 3 (TLR3) pattern recognition immune receptors (canonical activation ascribed to DNA and RNA, respectively; Schlee and Hartmann) to activate interferon regulatory transcription factor 3 (IRF3) signaling and induce cell death. Interestingly, the authors also noted hybrid excision and an R-loop-induced innate immune response in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (a rare autosomal recessive cerebellar ataxia associated with neurodegeneration; Suraweera et al.) and in human ovarian cancer cells containing mutated SETX and BRCA1 (Harding et al.).

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Conclusions, Questions, and Further Research

Overall, these data provide evidence that aberrant R-loop processing and the increase in the levels of cytoplasmic RNA–DNA hybrids induce an innate immune response that prompts cell death. Furthermore, the authors link this novel mechanism to conditions such as neurodegeneration and cancer. Future research may reveal the mechanisms underlying the regulation of R-loop processing and the passage of DNA-RNA hybrids from the nucleus to the cytoplasm.

For more on the molecular consequences of R-loop processing and links to disease, see Nature, December 2022.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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