Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity

Self- and non-self-nucleic acids are recognized by the innate immune system. Although RNA-DNA hybrids generated through R-loop formation during transcriptional stress have been implicated in innate immune activation, their physiological significance remains incompletely understood. Here, we demonstrate that nucleolar stress caused by dysfunction of nucleolar proteins RRN3 and ribosomal proteins, trigger the cytosolic accumulation of RNA-DNA hybrids originating from ribosomal DNA loci (ribo-RNA-DNA-hybrids). The formation of ribo-RNA-DNA-hybrids depend on XP proteins and partially activates cGAS- and TLR3-pathways, resulting in the activation of NF-κB without triggering IRF3 phosphorylation, leading to hyperinflammatory responses upon secondary stimulation. Importantly, cytosolic ribo-RNA-DNA hybrids increase in cells of Diamond-Blackfan anemia patients, certain cancer cells, myeloid cells stimulated with mRNA-LNP vaccines, correlating with heightened inflammatory responses. These findings reveal a crucial role for nucleolar stress-induced accumulation of cytosolic ribo-RNA-DNA hybrids under pathological conditions, including genetic disorders and tumorigenesis, as well as in the adjuvanticity of mRNA-LNP vaccines.