Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas

Molina, Rafael; Stella, Stefano; Mao, Feng; Sofos, N.; Jauniškis, Vykintas; Pozdnyakova, Irina; López-Méndez, Blanca; She, Qunxin; Montoya, Guillermo

doi:10.1038/s41467-019-12244-z

Cited by 79 publications

(99 citation statements)

References 53 publications

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“…We first wished to determine the identity of the activator for Csx1-Crn2 by incubating the enzyme with an RNA substrate (RNA A1) in the presence of cA3, cA4 or cA6. Only cA4 activated the ribonuclease activity of the enzyme (Figure 2A), consistent with the predicted structural similarity to the cA4-activated Csx1 protein of S. islandicus (10). A variant enzyme with a mutation targeted to the HEPN ribonuclease active site (H402A) was inactive for RNA degradation, but the H495A variant with a mutation targeted to the Crn2 active site appeared more active than the wild-type enzyme ( Figure 2B).…”

Section: The N-terminal Domain Of Csx1-crn2 Is a Ca4-activated Ribonusupporting

confidence: 78%

“…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%

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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: The N-terminal Domain Of Csx1-crn2 Is a Ca4-activated Ribonusupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

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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“…Next we asked whether the cOA moiety produced by SisCmr-β activates SisCsx1 as we previously demonstrated for cOA generated by SisCmr-α (Han et al, 2018;Molina et al, 2019). The reason for their co-existence is unclear.…”

Section: Introductionmentioning

confidence: 91%

Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

Sofos

Mao

Stella

et al. 2020

Preprint

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Cmr-β is a Type III-B CRISPR-Cas complex that upon target RNA recognition unleashes a multifaceted immune response against invading genetic elements, including ssDNA cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific ssRNA hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β unveiling how binding of the target RNA regulates the Cmr2 activities. CryoEM analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5´-handle in the crRNA and the 3´-antitag of the target RNA, activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self vs. non-self recognition. These findings highlight the diverse defense strategies of Type III complexes.

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“…RNases exhibit indiscriminate cleavage of viral and cellular RNAs, leading to cell dormancy or cell death to curb virus infection [38][39][40][41][42][43]50,51 . It is further believed that the type III CRISPR DNase eventually clears up remaining invading nucleic acids 52 , whereas the cOA secondary messenger is to be removed by ring nucleases 53 , allowing cells recover from the Type III immune response and restore the growth.…”

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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Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas

Cited by 79 publications

References 53 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

Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

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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