Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus

Abstract

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating “ring nuclease” activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.

The diverse arsenal of type III CRISPR–Cas-associated CARF and SAVED effectors

“…Cell death [72] N od a t a TIR-SAVED TIR-SAVED >3 cOA 3 [87] NAD + [87] Cell death [87] N od a t a Csa3 CARF-HTH 1 cOA 4 [53] dsDNA [53] Transcriptional regulation [53] No [53] Csx1…”

“…The complete regulatory functions of these effectors appear to be very complex, but hints at cross-talk between type III and type I systems. It has been shown that Csa3 is involved in the regulation of type I CRISPR adaptation, as well as providing a feedback loop to type III interference [50][51][52][53][54]. Furthermore, Csa3-mediated activation of DNA repair genes has been demonstrated, indicating that the network of gene regulation by type III associated effectors might not be constricted to CRISPR-related genes [55].…”

Structure of theSaccharolobus solfataricustype III-D CRISPR effector

“…These positions correspond to the sites of cleavage of bound target RNA (46), which is catalysed by the Cas7-like backbone subunits (47)(48)(49).This activity is important for the dissociation of target RNA and deactivation of the Cas10 subunit (44,45). Previous studies showed that SsoCsm is unusual in not cleaving at one of the flipped sites, generating a spacing of 12,6,6,6 between sites (42). The missed cleavage corresponds to the Cas7b subunit (1432; chain N in the PDB file, Figure 3C, D), while subunits competent for RNA cleavage are Cas7a (1430, chain L in the coordinates file), Cas7c (1425, chains F and G) and Cas7d (1426, chains H and I).…”

“…These ancillary nucleases typically have relaxed specificity and target both viral and host nucleic acids to slow down infection or cause abortive infection (11). The coordination of anti-viral responses at the transcriptional level by cA 4 are also possible (12).…”

Structure of the Saccharolobus solfataricus type III-D CRISPR effector

“…These ancillary nucleases typically have relaxed specificity and target both viral and host nucleic acids to slow down infection or cause abortive infection ( Rostol and Marraffini, 2019 ). The coordination of anti-viral responses at the transcriptional level by cA 4 are also possible ( Charbonneau et al., 2021 ).…”