Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA(4)) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA(4) and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA(4)-activated CRISPR ribonuclease (Csx1) and a cA(4)-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA(4) ligand(,) a canonical cA(4)-activated ribonuclease activity in the Csx1 domain and a potent cA(4) ring nuclease activity in the C-terminal Crn2 domain. The cA(4) binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA(4) levels as a second messenger of infection.