Nathaniel Burman

Cas10 is the signature gene for type III CRISPR–Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn(2+)-dependent deadenylation exoribonuclease. However, its specific role in CRISPR–Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA(4)) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA(4) signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR–Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA(4) signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

Abstract Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNA Lys . Phage T5 subverts PARIS immunity through expression of a tRNA Lys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells for diverse roles in antiviral defence. Here we determine structures of a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease and four molecules of a structure maintenance of chromosome (SMC)-family ATPase assemble into a 364 kDa complex that provides phage defence. We show that phage-encoded nucleases trigger degradation of the retron-associated DNA, leading to activation of the HNH nuclease. The HNH nuclease cleaves tRNASer, stalling protein synthesis and arresting viral replication. Taken together, these data reveal diverse and paradoxical roles for RTs in the perpetuation and elimination of genetic parasites. In this study, the authors determine the structure of a Type I-A retron from E. coli FORC82 and reveal the functional interplay between Reverse Transcriptases (RTs) and Structural Maintenance of Chromosomes (SMC) ATPases.’