Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains

The type III CRISPR-Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cA(n))- activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR-Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA(5) and cA(6). cA(6) acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm(6) domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cA(n)s to switch signaling off. CARF ring nuclease converts c(A)6 to linear A(6)>p and to the final A(3)>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA(6) or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Cs-m effector and the cA(6)-activated Csm6 ribonuclease.