Control of cyclic oligoadenylate synthesis in a type III CRISPR system

Rouillon, Christophe; Athukoralage, Januka S; Graham, Shirley A.; Grüschow, Sabine; White, Malcolm F.

doi:10.7554/elife.36734

Cited by 152 publications

(276 citation statements)

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“…In 1986, the presence of cAMP was reported in the archaeal species Methanobacterium thermautotrophicus , Sulfolobus solfataricus , and Haloferax volcanii (then referred to as Halobacterium volcanii ) (Leichtling, Rickenberg, Seely, Fahrney, & Pace, ), and cAMP levels have also been investigated in Halobacterium salinarum where a cell cycle‐dependent fluctuation of the intracellular cAMP concentration has been observed (Baumann, Lange, & Soppa, ). Another cyclic nucleotide that has been characterized in archaea is cyclic oligoadenylate, which has been shown to be involved in type III CRISPR‐mediated immunity in S. solfataricus (Rouillon, Athukoralage, Graham, Grüschow, & White, ). Comparative genome analyses have predicted different cyclases, including DACs, in additional archaeal species (Corrigan & Gründling, ; Shenroy & Visweswariah, ).…”

Section: Introductionmentioning

confidence: 99%

Cyclic nucleotides in archaea: Cyclic di‐AMP in the archaeonHaloferax volcaniiand its putative role

et al. 2019

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The role of cyclic nucleotides as second messengers for intracellular signal transduction has been well described in bacteria. One recently discovered bacterial second messenger is cyclic di‐adenylate monophosphate (c‐di‐AMP), which has been demonstrated to be essential in bacteria. Compared to bacteria, significantly less is known about second messengers in archaea. This study presents the first evidence of in vivo presence of c‐di‐AMP in an archaeon. The model organism Haloferax volcanii was demonstrated to produce c‐di‐AMP. Its genome encodes one diadenylate cyclase (DacZ) which was shown to produce c‐di‐AMP in vitro. Similar to bacteria, the dacZ gene is essential and homologous overexpression of DacZ leads to cell death, suggesting the need for tight regulation of c‐di‐AMP levels. Such tight regulation often indicates the control of important regulatory processes. A central target of c‐di‐AMP signaling in bacteria is cellular osmohomeostasis. The results presented here suggest a comparable function in H. volcanii. A strain with decreased c‐di‐AMP levels exhibited an increased cell area in hypo‐salt medium, implying impaired osmoregulation. In summary, this study expands the field of research on c‐di‐AMP and its physiological function to archaea and indicates that osmoregulation is likely to be a common function of c‐di‐AMP in bacteria and archaea.

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Section: Introductionmentioning

confidence: 99%

Cyclic nucleotides in archaea: Cyclic di‐AMP in the archaeonHaloferax volcaniiand its putative role

et al. 2019

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“…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.…”

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confidence: 99%

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

Athukoralage

McMahon

Zhang

et al. 2019

Preprint

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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 ...

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“…Gels were scanned by Typhoon FLA 7000 imager (GE Healthcare) at a wavelength of 532 nm, quantified using the Bio-Formats plugin 41 of ImageJ as distributed in the Fiji package 42 and plotted against the time using Kaleidagraph (Synergy Software, Reading, PA, United States). The data were fitted to a single exponential curve as previously described 1 .…”

Section: Can1 Nuclease Assaymentioning

confidence: 99%

“…Type III (Csm/Cmr) CRISPR systems utilise a cyclase domain in the Cas10 subunit to generate cyclic oligoadenylate (cOA) by polymerising ATP [1][2][3] . The cyclase activity is activated by target RNA binding and switched off by subsequent RNA cleavage and dissociation 1,4 . cOA is a potent anti-viral second messenger, that sculpts the anti-viral response by binding to and activating CRISPR associated Rossman fold (CARF) family proteins 5 .…”

Section: Introductionmentioning

confidence: 99%

“…The only CARF family enzymes to be characterised to date are dimeric ribonucleases of the Csx1/Csm6 family, which have a C-terminal HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) domain that is allosterically activated by cOA binding in the CARF domain [6][7][8][9] . Csm6 from Enterococcus italicus and Streptococcus thermophilus are activated by cyclic hexa-adenylate (cA6) 2,3 , whilst Csx1 from Sulfolobus solfataricus and Csm6 from Thermus thermophilus are activated by the cyclic tetraadenylate (cA4) molecule 1,2,10 . In addition to ribonucleases, a wide range of CARF family proteins have been identified, including transcription factors such as Csa3 11 , cA4-degrading ring nucleases 12 and putative membrane proteins of unknown function 5,13 , suggesting that much remains to be discovered.…”

Section: Introductionmentioning

confidence: 99%

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Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

McMahon

Zhu

Graham

et al. 2019

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these authors made equal contributions to the work presented. AbstractThe CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) molecules which act as a second messenger, signalling infection and potentiating a powerful immune response by activating a range of downstream effector proteins that can lead to viral clearance, cell dormancy or death. Only one type of effector enzyme has been studied -the Csm6/Csx1 ribonuclease domain, and the mechanism of cOA activation is not understood at a molecular level. Here we describe the structure and mechanism of a novel cOA-activated CRISPR defence DNA endonuclease, Can1 ("CRISPR ancillary nuclease 1"). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) CARF domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

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Control of cyclic oligoadenylate synthesis in a type III CRISPR system

Cited by 152 publications

References 42 publications

Cyclic nucleotides in archaea: Cyclic di‐AMP in the archaeonHaloferax volcaniiand its putative role

Cyclic nucleotides in archaea: Cyclic di‐AMP in the archaeonHaloferax volcaniiand its putative role

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

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

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