MDA5 guards against infection by surveying cellular RNA homeostasis

MDA5 is an innate immune RNA sensor that senses infection with a range of viruses and other pathogens. MDA5’s RNA agonists are not well defined. We used single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to study its ligands. Surprisingly, upon infection with SARS-CoV-2 or encephalomyocarditis virus, MDA5 bound overwhelmingly to cellular RNAs. Many binding sites were intronic and proximal to Alu elements and to potentially base-paired structures. Concomitantly, cytoplasmic levels of intron-containing unspliced transcripts increased in infected cells and displayed enrichment of MDA5 iCLIP peaks. Moreover, overexpression of a splicing factor abrogated MDA5 activation. Finally, when depleted of viral sequences, RNA extracted from infected cells still stimulated MDA5. Taken together, MDA5 surveys RNA processing fidelity and detects infections by sensing perturbations of posttranscriptional events such as splicing, establishing a paradigm of innate immune ‘guarding’ for RNA sensors.