Abstract
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 (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 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 cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 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 cA4 levels as a second messenger of infection.
Highlights
Type III CRISPR systems have class 1 effector complexes that utilize CRISPR RNA to detect RNA from mobile genetic elements (MGE) such as viruses
Type III CRISPR systems typically synthesize a range of cyclic oligoadenylate (cOA) molecules with a ring size varying from 3–6 [4,5,6,7,21]
Examples include cA4 and cA6 for type III CRISPR [5,6], cA3 for the HORMA-DncV-NucC system [24,25] and a variety of cyclic dinucleotides synthesised by diverse CBASS enzymes [26,27]
Summary
Type III CRISPR systems have class 1 effector complexes that utilize CRISPR RNA (crRNA) to detect RNA from mobile genetic elements (MGE) such as viruses This target RNA binding results in the activation of the Cas subunit, which commonly harbours two active sites: an HD nuclease domain for ssDNA cleavage [1,2,3] and a PALM polymerase domain that cyclises ATP to generate cyclic oligoadenylate (cOA) molecules [4,5,6,7]. Biochemical and modelling studies have revealed that AcrIII-1, which is widespread in archaeal viruses and bacteriophage, efficiently deactivates Csx in vitro [17]
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