Project Summary Human cells use signals called "RNA second messengers" to stimulate the immune response and prevent disease. These signals are produced in response to pathogen infection and cellular stress, and are important for controlling cellular communication. Recent evidence demonstrates that RNA second messenger signaling controlled by the enzyme cyclic GMP–AMP synthase (cGAS) is a critical component of the immune response to many types of cancer, and drug analogues developed from these natural signals are rapidly emerging as promising new treatments. The potent antitumor potential of these drugs illustrates the importance of discovery and mechanistic understanding of naturally occurring RNA second messenger signals. Surprisingly, our work has revealed that cGAS is part of a broad family of signaling enzymes we named cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes, and many RNA second messenger pathways remain to be discovered in human biology. Potential human CD-NTases include uncharacterized immune cGAS- like receptors previously implicated as genes frequently mutated in cancer. Our results demonstrate that RNA second messenger signaling pathways are a new rich area of research and likely impact many areas of basic human biology and disease. We have developed an innovative approach to uncover the function of these enzymes and discover the biology and natural products responsible for signaling. Our proposed research will provide a new detailed understanding of the enzymes that control RNA second messenger signaling and enable discovery of entirely new classes of RNA signals. Specifically, the proposed experiments will use kingdom-wide analysis of CD-NTase enzymes to determine the mechanism of specific RNA product synthesis, X-ray crystallography to define the structural and molecular basis of metazoan CD-NTase function, and an innovative set of cell biology experiments using newly identified CD-NTase RNA signals for direct cell stimulation and chemical-proteomics. Our experiments will define the function of newly discovered cGAS-like enzymes in human biology and explain the molecular rules that allow RNA second messengers to control downstream cellular responses.