Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

McMahon

Zhang

et al. 2019

Preprint

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger 1-3 , activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction 4,5 . Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 6,7 . Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.Type III CRISPR-Cas systems synthesise the signalling molecule cyclic oligoadenylate (cOA) from ATP 1,2 when they detect viral RNA. cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain. CARF domains are found fused to a variety of effector domains that are known or predicted to cleave RNA, DNA, or function as transcription factors 11 . The best characterised CARF protein family is the Csx1/Csm6 family of HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) ribonucleases, which are activated by cOA binding and cleave RNA with minimal sequence dependence 1-3 . A number of studies have demonstrated that the cOA signalling component of type III systems is crucial for effective immunity against viruses 4,12-15 , highlighting the importance of this facet of CRISPR immunity.Recently, we identified a cellular enzyme in Sulfolobus solfataricus, hereafter referred to as the Crn1 family (for "CRISPR associated ring nuclease 1"), that degrades cA4 molecules and thus deactivates the Csx1 ribonuclease in vitro 16 . These enzymes exhibit very slow kinetics, and are thought to act by mopping up cA4 molecules in the cell without compromising the immunity provided by the type III CRISPR system. Unsurprisingly, viruses have responded to the threat of the CRISPR system by evolving a range of anti-CRISPR (Acr) proteins, which are used to inhibit and overcome the cell's CRISPR defences (reviewed in 17 ). Acr's have been identified for the type I-D 18 , I-F , II-A and V-A effector complexes (reviewed in 17,19,20 ), numbering over 40 families 21 , but importantly not for type III systems. We focussed on one of the protein families, DUF1874, conserved and widespread ...

mentioning

confidence: 99%

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

McMahon

Zhang

et al. 2019

Preprint

“…A significant increase in the amounts of the larger cA6 and cA5 species was observed, with cA6 coming to predominate. This reflects the finding that target RNA cleavage by the Csm3 subunit is the event that deactivates the cyclase domain (13,40). However, even with target RNA cleavage abolished, a range of cOA species are synthesised.…”

Section: Discussionmentioning

confidence: 56%

“…Although only type III-A systems are known to utilize cA6, there are well-studied examples (such as T. thermophilus and M. thermautotrophicus) of type III-A systems with ancillary ribonucleases activated by cA4. All type III systems studied to date have the capability of making a range of cyclic oligoadenylates from cA3-cA6 (11)(12)(13)40). As we have shown here, the immunity provided by a type III system can be readily converted from a cA6 to a cA4-based mechanism, simply by replacing the ancillary enzyme.…”

Section: Discussionmentioning

confidence: 85%

Cyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence

Grüschow

Graham

et al. 2019

Preprint

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE is achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show that the mechanism can be reprogrammed to operate as a cyclic tetra-adenylate (cA4) system by replacing the effector protein. These observations demonstrate that M. tuberculosis has a fullyfunctioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

“…Type III CRISPR systems have class 1 effector complexes that utilize CRISPR RNA (crRNA) to detect invading RNA from mobile genetic elements (MGE) such as viruses. This target RNA binding results in the activation of the Cas10 subunit, which commonly harbours two active sites: an HD nuclease domain for ssDNA cleavage (1-3) and a PALM polymerase domain that cyclises ATP to generate cyclic oligoadenylate (cOA) molecules (4)(5)(6)(7). cOA acts as a second messenger in the cell, signalling infection and activating a range of auxiliary defence enzymes such as the ribonuclease Csx1/Csm6 (8)(9)(10) or the DNA nickase Can1 (11) by binding to a CRISPR-associated Rossman fold (CARF) sensing domain.…”

Section: Introductionmentioning

confidence: 99%

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

Samolygo

Graham

et al. 2020

Preprint

AbstractType 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. To avoid this fate, 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 activities of the two constituent enzymes in the fusion protein cooperate to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.