Journal: Nucleic Acids Research

Article Title: Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR–Cas immunity

doi: 10.1093/nar/gkaa176

Figure Lengend Snippet: Model for regulation of the RNAse, DNase, and cOA synthetase activities of the Pfu Type III-B effector crRNP in providing anti-viral immunity. Inactive Type III-B crRNPs become activated upon interaction with expressed viral RNA. crRNA-guided base-pairing to the viral RNA and Cas protein recognition of the PFS results in conformational changes that trigger three important activities: viral RNA cleavage by Cmr4, viral DNA cleavage by the HD domain of Cmr2 (a member of the Cas10 superfamily), and ATP dependent production of cyclic oligoadenylates (cOA) by the Palm domain of Cmr2. The cOA second messengers allosterically regulate the trans-acting Csx1 enzyme by binding to the CARF domain to trigger HEPN RNase activity. Clearance of the viral DNAs and RNAs provides immunity and restores the crRNPs back to the inactive state. Under conditions of controlled immunity, Csx1 degrades and inactivates cOA signaling molecules using the HEPN domain, to provide an auto-inhibitory off-switch of Csx1 RNase activation. Inefficient degradation of cOA molecules by the Csx1 or failure to efficiently destroy the viral RNAs or viral genome may cause Csx1 to destroy both viral and cellular RNA molecules, leading to cellular dormancy or death.

Article Snippet: Pyrococcus furiosus ( Pfu ) is a hyperthermophilic archaeon that contains three distinct functional CRISPR–Cas effector crRNPs: two DNA-targeting systems (Types I-A (Csa) and I-B (formerly known as I-G) (Cst) ( )) and a Type III-B (Cmr) system ( ) shown to destroy both DNA and RNA targets in a transcription-coupled manner ( , , , ).

Techniques: Binding Assay, Activity Assay, Activation Assay