Regulation of cGAS activity by RNA‐modulated phase separation

Cyclic GMP‐AMP synthase (cGAS) is a double‐stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA, cGAS and dsDNA form phase‐separated condensates in which cGAS catalyzes the synthesis of 2′3′‐cyclic GMP‐AMP that subsequently triggers a STING‐dependent, type I interferon (IFN‐I) response. Here, we show that cytoplasmic RNAs regulate cGAS activity. We discover that RNAs do not activate cGAS but rather promote phase separation of cGAS in vitro . In cells, cGAS colocalizes with RNA and forms complexes with RNA. In the presence of cytoplasmic dsDNA, RNAs colocalize with phase‐separated condensates of cGAS and dsDNA. Further in vitro assays showed that RNAs promote the formation of cGAS‐containing phase separations and enhance cGAS activity when the dsDNA concentration is low. Cotransfection of RNA with a small amount of dsDNA into THP1 cells significantly enhances the production of the downstream signaling molecule interferon beta (IFNB). This enhancement can be blocked by a cGAS‐specific inhibitor. Thus, cytoplasmic RNAs could regulate cGAS activity by modulating the formation of cGAS‐containing condensates. Synopsis cGAS‐dsDNA phase separation is critical for the dsDNA‐induced activation of cGAS. This study shows that RNA‐modulated phase separation of cGAS plays a role in regulating cGAS activity. RNA does not directly activate cGAS but modulates the formation of cGAS‐dsDNA condensates. RNA promotes phase separation of cGAS‐dsDNA and enhances cGAS activity at low dsDNA concentrations. RNA competes with dsDNA in phase‐separated granules of cGAS and inhibits cGAS activity at high dsDNA concentrations. Graphical Abstract cGAS‐dsDNA phase separation is critical for the dsDNA‐induced activation of cGAS. This study shows that RNA‐modulated phase separation of cGAS plays a role in regulating cGAS activity.