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

Funding: Biotechnology and Biological Sciences Research Council [REF BB/S000313/1 to M.F.W.]; Funding for open access charge: RCUK block grant.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.Publisher PDFPeer reviewe

Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.Publisher PDFPeer reviewe

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (Grant REF BB/S000313/1 to MFW) and the Wellcome Trust (Grant 210486/Z/18/Z to CMC).Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.Publisher PDFPeer reviewe

Killing the messenger : discovery of enzymes degrading cyclic oligoadenylate defence activators synthesised by type III CRISPR immune systems

CRISPR-Cas systems provide prokaryotes with adaptative immunity from invading Mobile Genetic Elements (MGEs). Type III CRISPR-Cas systems consist of a multiprotein effector complex and CRISPR ancillary proteins; both essential for MGE elimination. In 2017, two groups independently identified that type III CRISPR-Cas complexes synthesised cyclic oligoadenylate (cOA) second messengers in response to detection of foreign RNA. cOA was made using ATP (adenosine triphosphate) and consisted of 3-6, 3’-5’ linked AMP subunits (cAn, n=3-6). cOA was found to activate CRISPR ancillary nucleases, which eliminated MGEs by cleaving nucleic acids non- specifically. CRISPR ancillary proteins are diverse, consisting of a cOA sensor domain fused to a toxin effector domain. This led to speculation that, if not controlled, collateral damage from the immune response could lead to cell dormancy or death, an unfavourable outcome for unicellular organisms. The work described herein details the identification of a new class of enzyme, termed “ring nuclease”, which degrades cyclic tetra-adenylate (cA4) second messengers and regulates the type III CRISPR immune response. The publications presented characterise five distinct ring nuclease families. These include the CRISPR ring nuclease 1 (Crn1) limited to the archaea, cOA activated self-inactivating CRISPR ancillary ribonucleases and the highly unusual Csx3/Crn3 family, which collectively extend ring nuclease distribution. Also presented are the anti-CRISPR DUF1874 family variant ring nucleases, which viruses employ to subvert type III CRISPR immunity, and the homologous Crn2 family found in association with type III CRISPR-Cas systems in prokaryotic genomes. Biochemical and biophysical characterisation of these proteins reveal diverse mechanisms underlying regulation of type III CRISPR defence, and kinetic modelling demonstrate the key roles of ring nucleases in governing the outcome of infections. These works provide fundamental insights into the regulation of a sophisticated, widespread and potent prokaryotic immune system, and select ring nucleases hold great promise for increasing the efficacy of bacteriophage therapies targeting pathogenic bacteria

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

This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/S000313/1 to M.F.W. and BB/R008035/1 to T.M.G.) and by a NASA Exobiology and Evolutionary Biology grant (NNX14AK23G to R.J.W.).The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3,4,5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7,8,9,10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host–pathogen interactions11,12,13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize 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. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.PostprintPeer reviewe

Activation of Csm6 ribonuclease by cyclic nucleotide by cyclic nucleotide binding : in an emergency twist to open

Biotechnology and Biological Sciences Research Council [BB/T004789/1 to M.F.W. and T.M.G.]; European Research Council [101018608 to M.F.W.]; equipment was funded by BBSRC [BB/R013780/1 and BB/T017740/1]; T.M.G. is a recipient of a Royal Society Leverhulme Trust Senior Research Fellowship [SRF\R1\221056].Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6’ ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6’ in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the ‘jaws’ of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.Publisher PDFPeer reviewe