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

Figure 1.. Csx1 is required for anti-plasmid immunity in the P. furiosus (Pfu) III-B CRISPR–Cas system. (A) Components of the Pfu Cmr defense response. The Cmr effector complex is composed of Cmr1–6 and a mature crRNA (black & orange) containing a 5′ tag (black) eight nucleotides in length. Cyclic oligoadenylate compounds (orange) produced by the Palm domain of Cmr2 bind to the CARF domain of Csx1. See Supplemental Figure S1 for a list of the Cas gene family names that correspond to each of the six Cmr subunits. (B) Plasmid interference assay. Pfu strains were transformed with plasmids that contain (target) or lack (no target) a transcribed target region complementary to the 7.01 crRNA (first spacer of the Pfu CRISPR 7 array). Both plasmids contain the pyrF gene to facilitate growth in the absence of uracil. (C) Colonies produced by transforming 11 different Pfu strains. Strains were transformed with the target plasmid (blue) or no target plasmid (gray). Mean colonies obtained per μg of plasmid DNA are plotted for two replicates. Error bars indicate standard deviation of replicates.

Figure 2.. The ribonuclease activity of Pfu Csx1 is activated by cOA species produced by the Pfu effector complex in a Cmr2 Palm domain-dependent manner. (A) Purification of Pfu complex proteins. HD and Palm mutations are located within Cmr2. The D26N mutation is located within Cmr4. (B) RNase activity of each Pfu Cmr complex. Radiolabeled 7.01 Target RNA was incubated with Cmr complexes and reaction products were visualized by urea-PAGE. Radiolabeled RNA size standards (M) in nucleotides were used. The black arrow indicates the full-length substrate and the black asterisks indicate cleavage products. (C) DNase activity of each Pfu Cmr complex. Each complex was incubated with dsDNA (label located on DNA Target 2) and reactions were visualized as described in part B. (D) cOA production activity of the Pfu Cmr crRNP. The five Cmr complexes were tested for cOA production in presence (+) and absence (–) of 7.01 Target RNA. Reaction products were run on a denaturing gel. A radiolabeled alkaline hydrolysis ladder (M2) and size standard (M) were added to the urea-PAGE analysis. (E) Activation of wildtype Csx1 by Pfu cOAs. Csx1 was tested for ribonuclease activity under activating (20 nM Csx1)…

Figure 3.. cA4 is the relevant activator for Pfu Csx1. (A) UV chromatogram (258 nm) and MS chromatogram (extracted ion chromatogram for m/z 494.6, 659.1, 823.6, 988.2) for cA samples from Pfu Cmr wildtype complexes. (B) Pfu Csx1 activation assays with synthetic cOA. Wildtype Csx1 (20 nM) was incubated with increasing concentrations of synthetic cA4 and cA6. For each activator, 1.85, 18.5 and 185 pM concentrations were used. A radiolabeled alkaline RNA size marker (M) was added to the urea-PAGE analysis. The black arrow indicates the full-length RNA substrate and the black asterisks indicate cleavage products. Dotted lines are indicative of noncontiguous data being omitted from the gel.

Figure 4.. Activation of Pfu Csx1 is CARF domain dependent. (A) Ribbon structure of Pfu Csx1 (PDB: 4EOG) indicating the CARF and HEPN domains. Residue H436 was mutated in the HEPN domain (green). Residues 121–127 were deleted from the CARF domain (blue), and residues I169 and N170 were mutated within the CARF domain (red). (B) SDS-PAGE analysis of Pfu Csx1 protein purifications. WTcolumn refers to protein purified using a column method and WTbatch refers to protein purified using a batch method. (C–E) In vitro activation of Csx1 mutants. Wildtype Csx1 along with four Csx1 mutants (CARFm, INAA, INQQ, HEPNm) was tested for ribonuclease activity under high (500 nM Csx1) and low (10 nM Csx1) concentration conditions. A radiolabeled alkaline RNA size marker (M) was added to the urea-PAGE analysis. The black arrow indicates the full length RNA substrate and the black asterisks indicate cleavage products. (F) In vivo plasmid silencing assay results for CARF domain mutants. Strains were transformed with the target plasmid (blue) or no target plasmid (gray). Mean colonies obtained per μg of plasmid DNA are plotted for two replicates. Error bars indicate standard deviation of replicates.

Figure 5.. Degradation of cOAs by Pfu Csx1 inactivates the cOA activators. (A) Sequencing gel reactions of Csx1 proteins incubated with Pfu cOAs. Five different Pfu Csx1 proteins were incubated with radiolabeled cOAs for two timepoints-5 and 20 mins. Urea-page analysis on sequencing gels was performed. Radiolabeled RNA size marker (M) and alkaline hydrolysis ladder (M) and were included in the analysis. (B) Effect of Csx1 treatment on cOA activation activity. Unlabeled Pfu cOAs were treated with 600 nM of wildtype Csx1 or no protein for 30 min. The plus extraction set of reactions were tested for activation of wildtype Csx1 by incubating 20 nM of wildtype Csx1 with decreasing concentrations of A2>P¥ or cOA4— roughly 1.25, 0.6, 0.6 × 10−1, 0.6 × 10−2, and 0.6 × 10−4 uM. ¥ indicates a putative designation for this reaction product. Native cOA without the 30 min incubation or extraction was utilized as a control (N).

Figure 6.. Pfu Cmr DNase and cOA production activities are PFS dependent. (A) Composition of the Pfu Cmr crRNP. The Cmr effector complex is composed of Cmr1–6 and a mature crRNA (black & orange) containing a 5′ tag (black). The Pfu Cmr complex is activated by binding of complementary target RNA (purple) to the crRNA. The target RNA contains a PFS (red) within it that is located adjacent (3′) to the target sequence. (B) Target RNA dependent activation of DNase activity by the Pfu complex. Cmr crRNP complexes were incubated with radiolabeled double-stranded DNA in the presence of 23 different target RNAs. Each target RNA contains a different three nucleotide sequence in the PFS region of the target RNA. Urea-page analysis on sequencing gels was performed. Radiolabeled RNA size markers (M) were included in the analysis. The black arrow indicates the full-length RNA substrate and the black asterisks indicate cleavage products. (C) Target RNA dependent activation of cOA production activity by the Pfu complex using the same target RNAs mentioned in part B. Products were analyzed as described in part B. An alkaline hydrolysis ladder (M2) was included in the urea-PAGE analysis.

Figure 7.. 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…