Cyclic oligoadenylate signalling and regulation by ring nucleases during type III CRISPR defence

Abstract

In prokaryotes, CRISPR-Cas immune systems recognise and cleave foreign nucleic acids to defend against Mobile Genetic Elements (MGEs). Type III CRISPR-Cas complexes also synthesise cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defence. In particular, cOA-stimulated nucleases degrade RNA and DNA non-specifically, which slows MGE replication but also impedes cell growth, necessitating mechanisms to eliminate cOA in order to mitigate collateral damage. Extant cOA is degraded by a new class of enzyme termed a 'ring nuclease', which cleaves cOA specifically and switches off CRISPR ancillary enzymes. Several ring nuclease families have been characterised to date, including a family used by MGEs to circumvent CRISPR immunity, and encompass diverse protein folds and distinct cOA cleavage mechanisms. In this review we outline cOA signalling, discuss how different ring nucleases regulate the cOA signalling pathway, and reflect on parallels between cyclic nucleotide-based immune systems to reveal new areas for exploration.

Keywords: CARF; CRISPR-Cas; Csm6 ribonuclease; cyclic nucleotides; ring nuclease.

FIGURE 1.

Cyclic oligoadenylate signaling by type III CRISPR systems. Multisubunit type III CRISPR-Cas complexes (denoted Csm or Cmr) detect foreign RNA and carry out nucleic acid cleavage directly and by recruiting CRISPR ancillary enzymes. RNA bound by the CRISPR-Cas complex, as a result of complementary base-pairing with the crRNA, is degraded by the Cas7 (Csm3/Cmr4) backbone subunits (Benda et al. 2014; Ramia et al. 2014; Staals et al. 2014; Tamulaitis et al. 2014). Bona fide RNA targets contain a 3′-region that is not complementary to the 8 nt 5′-end of the crRNA, which allosterically activates the Cas10 subunit to synthesize cyclic oligoadenylates (cOA) and cleave ssDNA (Kazlauskiene et al. 2017; Niewoehner et al. 2017). Target RNA cleavage by Cas7 subunits switches off both the ssDNase and cOA synthesis activities of Cas10 (Rouillon et al. 2018). cOA can activate Csx1/Csm6 ribonucleases that cleave RNA nonspecifically, DNases such as NucC (Lau et al. 2020) and the CRISPR ancillary nuclease 1 (Can1) (McMahon et al. 2020), and the related dual-specificity cOA-activated RNase and DNase (Card1)/Can2 (Rostøl et al. 2021; Zhu et al. 2021), which help eliminate invading mobile genetic elements (MGE). cOA can also stimulate the transcription regulator Csa3, which alters CRISPR loci and cas gene expression to promote MGE elimination (Lawrence et al. 2020).

FIGURE 2.

Structures of type III CRISPR ancillary proteins. (A) E. italicus (Eit) Csm6 dimer in complex with its cyclic hexa-adenylate (cA₆) activator (shown in sphere form). EitCsm6 is a nonspecific ribonuclease containing CARF (dark and light blue) and HEPN (dark and light green) domains (Garcia-Doval et al. 2020). (B) T. onnurineus (Ton) Csm6 dimer in complex with its cyclic tetra-adenylate (cA₄) activator. TonCsm6 is a nonspecific ribonuclease consisting of CARF and HEPN domains (Jia et al. 2019c). (C) S. islandicus (Sis) Csx1 hexamer in complex with its cA₄ activator. SisCsx1 dimers form a hexamer upon cA₄ binding and RNA is cleaved at three distinct active sites within the interior of the hexamer (Molina et al. 2019). (D) S. solfataricus (Sso) Csa3 dimer. SsoCsa3 is a cA₄ stimulated transcription regulator consisting of CARF and a helix-turn-helix DNA binding domain (yellow and orange) (Lintner et al. 2011). (E) T. thermophilus (Tth) CRISPR ancillary nuclease 1 (Can1) monomer in complex with its cA₄ activator. TthCan1 nicks super-coiled DNA and is comprised of two CARF domains and a PD-D/ExK family nuclease domain (salmon colored) (McMahon et al. 2020). (F) T. succinifaciens cyclic oligoadenylate activated RNase and DNase 1 (Card1)/Can2 dimer in complex with its cA₄ activator. Card1/Can2 is related to Can1 and is a dual-specificity nuclease with CARF and PD-D/ExK nuclease domains (red and salmon) (Rostøl et al. 2021; Zhu et al. 2021). (G) E. coli (Eco) NucC hexamer in complex with its cyclic tri-adenylate (cA₃) activator. EcoNucC trimers assemble into a hexamer upon cA₃ binding and degrades dsDNA (Lau et al. 2020). NucC is related to restriction endonucleases and binds cA₃ at a protein domain unrelated to the CARF family.

FIGURE 3.

Ring nucleases regulate the type III CRISPR immune response. (A) Cyclic tetra-adenylate (cA₄)-activated Csx1 ribonucleases cleave RNA nonspecifically, which provides antiviral immunity but also causes collateral damage to cells. cA₄ binds the CRISPR-associated Rossmann fold (CARF) domain of Csx1 and allosterically activates its higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain, which cleaves RNA. CRISPR ring nucleases 1 and 3 eliminate extant cA₄ and deactivate Csx1, mitigating sustained collateral damage to cells. (B) Graph depicting kinetic modeling of the type III CRISPR response in a cell where 60 µM of cA₄ is generated upon infection, representative of a medium-level infection. In the absence of ring nucleases, Csx1 remains in an active state (dotted green line, corresponding to left-hand side y-axis) and RNA cleavage (solid green line, corresponding to right-hand side y-axis) continues unimpeded. Csx1 is slowly deactivated when CRISPR ring nuclease 1 is present (dotted blue line) and thus RNA cleavage is limited (solid blue line). Simulations were carried out using KinTek Global Kinetic Explorer software (Johnson et al. 2009), using a previously published model of the S. solfataricus type III CRISPR defense pathway (Athukoralage et al. 2020a), and data were plotted using GraphPad Prism. (C) Some Csm6 enzymes act as bifunctional ribonucleases and ring nucleases. These enzymes cleave cA₄ at the CARF domain upon cA₄ binding and activating the HEPN RNase. Some Csm6 enzymes also cleave cOA at the HEPN domain. (D) Prokaryotic viruses encode an anti-CRISPR viral ring nuclease (AcrIII-1). AcrIII-1 rapidly degrades cA₄ and attenuates RNA cleavage by swiftly deactivating Csx1. (E) Graph depicting the effect of having no ring nuclease (green), a host cell CRISPR ring nuclease 1 (blue), and both CRISPR ring nuclease 1 and AcrIII-1 on the active form of Csx1 (dotted lines, left-hand side y-axis) and consequent RNA cleavage (solid lines, right-hand side y-axis). When AcrIII-1 is present, Csx1 is deactivated much more quickly. (F) In some bacteria, AcrIII-1 homologs are found associated with type III CRISPR systems and these proteins have been named CRISPR ring nuclease 2 (Crn2). In Marinitoga piezophile, Csx1 is fused to Crn2, which limits Csx1 activity by rapidly and constitutively degrading cA₄. The Crn2 domain only permits Csx1 activation when a high cA₄ threshold, determined by the balance between cA₄ affinity of the CARF domain of Csx1 and the high cA₄ affinity and rate of degradation by Crn2, is reached.

FIGURE 4.

Comparisons of cyclic nucleotide-based immune systems in prokaryotes. (A) Protein Data Bank (PDB) identifiers are shown alongside all structures. Type III CRISPR immunity comprises four key components; a detection and signaling platform, cyclic nucleotide signals, ancillary signal sensors fused to effectors, and mechanisms to eliminate the signal. The type III CRISPR complex detects viral mRNA, and the Cas10 nucleotidyl cyclase generates cyclic oligoadenylates (cOA; cA_n, n = 3–6) containing 3′–5′ phosphodiester linkages. cOA allosterically stimulates downstream CRISPR ancillary effector proteins, typically by binding to a CRISPR-associated Rossmann fold (CARF) domain (colored in marine and light blue in protein dimers). Finally, ring nucleases eliminate cOA and deactivate CRISPR ancillary effectors, controlling the immune response. Some ring nucleases are CARF family proteins, whereas others have unique cOA sensing domains. Viruses encode variant ring nucleases (AcrIII-1), which rapidly degrade cOA and suppress the type III CRISPR immune response. In some bacteria, AcrIII-1 is found associated with type III CRISPR systems and has been termed Crn2 because it appears to be harnessed by bacteria to regulate CRISPR immunity. (B) The cyclic oligonucleotide-based antiphage signaling system (CBASS) resembles the type III CRISPR immune system in some respects. The cyclase (CdnE) is activated by unknown stimuli during phage infection, and different CdnE proteins synthesize different cyclic nucleotide molecules which activate downstream effector proteins. CBASS can synthesize cyclic nucleotides containing both 3′–5′ and 2′–5′ phosphodiester linkages, giving rise to an enormous repertoire of possible cyclic nucleotide signals. These signals are detected by distinct protein domains, including the SMODS associated and fused to various effectors sensor domain (SAVED), which is evolutionarily linked to the CARF domain. Although ring nucleases may degrade cA₄, no prokaryotic enzymes have been identified that degrade any of the other cyclic nucleotide signals generated by CBASS. Some CBASS systems may function exclusively via abortive infection, whereas others may have novel regulatory mechanisms.