A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator

This work was funded by a grant from the Biotechnology and Biological Sciences Research Council (Grant REF BB/S000313/1 to MFW).Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.Publisher PDFPeer reviewe