RIG-I-like receptors sense replication stress through ribosomal DNA integrity to drive inflammation

Replication stress, a major source of DNA damage and vulnerability in cancer, activates innate immunity through both the cGAS–STING and RIG-I–like receptor (RLR) pathways, yet the mechanism for RLR activation remains poorly understood despite frequent suppression of cGAS–STING in cancer. Here, we show that replication stress induces genomic instability at ribosomal DNA (rDNA), accompanied by aberrant RNA polymerase I–dependent sense and antisense transcription that generates immunostimulatory RNA activating RLR signaling. RLR activation required CDK1 activity but occurred independently of mitosis or micronuclei formation. Cas9-mediated disruption of transcribed rDNA regions was sufficient to induce dysregulated rDNA transcription and RLR activation. Human tumor genomes also showed increased structural variations at rDNA, consistent with rDNA instability. Together, these findings reveal how genomic instability drives inflammation through rDNA-derived immunostimulatory RNA and identify a previously unappreciated consequence of damage-induced transcription.