CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA&gt;p Formation Terminating RNase Activity

Jia, Ning; Jones, R. A. C.; Yang, Guangli; Ouerfelli, Ouathek; Patel, Dinshaw J.

doi:10.1016/j.molcel.2019.06.014

Cited by 94 publications

(112 citation statements)

References 45 publications

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“…For type III CRISPR systems at least, immunity rather than abortive infection may be the modus operandi, as mechanisms exist to "kill the messenger". Ring nucleases that degrade cA4 include the dedicated enzymes and self-limiting ribonucleases based on the CARF domain (9,14,15) and the DUF1874 family whose members include the viral anti-CRISPR AcrIII-1 and putative cellular Crn2 proteins (16).…”

Section: Discussionmentioning

confidence: 99%

“…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. Once activated, these enzymes degrade both host and invading nucleic acids in the cell, which can result in viral clearance (i.e.…”

Section: Introductionmentioning

confidence: 99%

“…In S. solfataricus, cells express a specialised catalytic variant of a CARF-domain protein named "CRISPR-associated ring nuclease 1" (Crn1) that binds and slowly degrades cA4, returning cells to a basal, uninfected state (14). The CARF domains of some members of the Csm6 family have also been shown to have ring nuclease activity, slowly degrading cA4 and thus acting as self-limiting ribonucleases (9,15). More recently, a potent ring nuclease enzyme of viral origin has been identified that acts as an anti-CRISPR (Acr) by rapidly degrading cA4 to circumvent type III CRISPR immunity (16).…”

Section: Introductionmentioning

confidence: 99%

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

Samolygo

Athukoralage

Graham

et al. 2020

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Samolygo

Athukoralage

Graham

et al. 2020

Preprint

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“…The Crn1 family (represented here by Sso1393 PDB code 3QYF) is restricted to the crenarchaea . The Csm6/Csx1 family (represented here by PDB code 6O6Y) are self-limiting cA 4 -dependent ribonucleases , Jia et al, 2019, Garcia-Doval et al, 2020. The Crn3 family is present in type III CRISPR systems in euryarchaeal and cyanobacteria.…”

Section: Figure 6 Sigmoidal Response Of Ring Nuclease Activity As a mentioning

confidence: 99%

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Graham

Grüschow

Gloster

et al. 2020

Preprint

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9Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to 11 signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This 12 can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell 13once a viral infection has been defeated. Enzymes specialised for this task are known as ring 14nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR 15 associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that 16rapidly degrades cyclic tetra-adenylate (cA 4 ). The enzyme has an unusual cooperative reaction 17 mechanism involving an active site that spans the interface between two dimers, sandwiching the 18 cA 4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 19family. 20 Keywords: 21 CRISPR, Csx3, ring nuclease, cyclic tetra-adenylate, CARF, cooperative enzyme 22 103 terminal Csx3 domain fused to a C-terminal kinase/transferase domain of unknown function. B. Phosphor 104 images of native gel electrophoresis visualising cA 4 (20 nM) or RNA oligonucleotide 49-9A (50 nM) binding 105 by A. fulgidus Csx3. Csx3 binds to cA 4 with high affinity (apparent K D ~ 50 nM) and binds the RNA 49-9A 106 with significantly lower affinity (apparent K D ~ 10 µM). Images are representative of three technical 107 replicates. 108 109 Figure 1figure supplement 1. Multiple sequence alignment of Csx3 proteins showing conserved 110 residues. Csx3 proteins are shown from Archaeoglobus fulgidus; Methanosarcinia mazei; candidatus 111 Bathyarchaeota; Thermococcus celericrescens; Methylacidiphilum fumariolicum; Aquifex aeolicus; 112 Dictyoglomus turgidum; Spirulina major; Oscillatoria nigro-viridis. Absolutely conserved residues are shaded. 113 Conserved residues H60 and D69 (Afu numbering) are indicated by asterisks. 114 131 Figure 2. Csx3 is a potent ring nuclease. A. TLC analysis of the reaction products of radiolabelled cA 4 132 (200 nM) incubated with A. fulgidus Csx3 (8 µM dimer) in reaction buffer at 50 °C (time points every 5 s from 133 10-60 s then 1.5, 2, 3, 4, 5 and 10 min, n = 6 technical replicates). The cA 4 was rapidly converted into A 2 -P, 134 with a small amount of A 2 >P (linear A 2 with a cyclic 2',3' terminal phosphate). B. Denaturing polyacrylamide 135 gel electrophoresis visualising cleavage of 5'-end radiolabelled RNA 49-9A (50 nM) by Csx3 (8 µM dimer) at 136 50 °C (time points every 5 min from 5-40 min then 50, 60 and 90 min, n = 3 technical replicates). C. Plot 137 comparing single-turnover kinetics of cA 4 (blue) and RNA 49-9A (green) cleavage by AfCsx3 at 50 °C. Data 138 points are the average of three technical replicates and error bars represent the standard deviation of the 139 mean. D. Liquid-chromatography high-resolution mass spectrometry analysis of reaction products when cA 4

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“…Type III CRISPR systems are unique because they exhibit both RNA interference and DNA interference in vivo to protect their microbial hosts against invading nucleic acids 4,[12][13][14][15][16][17][18][19][20][21][22][23] . Three activities are associated with these Type III immune systems, including target RNA cleavage 18,[24][25][26][27][28][29][30] , target RNAactivated indiscriminate single-stranded (ss) DNA cleavage, a secondary DNase activity 15,19,20,[31][32][33][34][35][36][37] and synthesis of cyclic oligoadenylate (cOA), a second messenger that activates RNases of Csm6/Csx1 families, mediating cell dormancy or cell death [38][39][40][41][42][43][44][45] . Activation of the immunity requires mismatches between the 3ʹ anti-tag of a cognate target RNA (CTR) and the 5ʹ tag of crRNA whereas the full match between the 3ʹ anti-tag of non-cognate target RNA (NTR) and the 5ʹ tag of crRNA completely represses the immune response (see reviews [46][47][48][49].…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

Lin

Mao

Zhang

et al. 2019

Preprint

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AbstractAntiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in Escherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3′ anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3′ anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match in the noncognate target RNAs (NTR), to the 5’ tag of crRNA. In addition, further increasing the number of 3′ anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.

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CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA>p Formation Terminating RNase Activity

Cited by 94 publications

References 45 publications

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

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

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

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