Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, United Kingdom
Figures
Figure 1 with 1 supplement
Csx3 is a type III CRISPR accessory protein that binds cA4.
(A) Genome context of selected Csx3 orthologues. Csx3 is found next to type III-B CRISPR operons and adjacent to Csx1 and other uncharacterised CARF family proteins. Csx3’ is a longer version of …
Figure 1—figure supplement 1
Multiple sequence alignment of Csx3 proteins showing conserved residues.
Csx3 proteins are shown from Archaeoglobus fulgidus; Methanosarcinia mazei; Candidatus Bathyarchaeota; Thermococcus celericrescens; Methylacidiphilum fumariolicum; Aquifex aeolicus; Dictyoglomus …
Figure 2 with 1 supplement
Csx3 is a potent ring nuclease.
(A) Denaturing polyacrylamide gel electrophoresis visualising cleavage of 5’-end radiolabelled RNA 49-9A (50 nM) by Csx3 (8 µM dimer) at 50°C (time points every 5 min from 5 to 40 min, then 50, 60 …
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Figure 2—source data 1
Excel spreadsheet with raw data.
- https://cdn.elifesciences.org/articles/57627/elife-57627-fig2-data1-v2.xlsx
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Figure 2—source data 2
Excel spreadsheet with raw data.
- https://cdn.elifesciences.org/articles/57627/elife-57627-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Csx3 from M. mazei is a ring nuclease.
TLC analysis of cleavage products obtained after incubating radiolabelled cA4 (~200 nM) with MmCsx3 (8 µM dimer) in reaction buffer at 50°C (time points are 10 s, 20 s, 30 s, 1 min, 5 min, 15 min, …
Figure 3
Csx3 functions as a ring nuclease in vivo.
(A) Schematic of plasmid transformation assay. A plasmid containing the effector nuclease Csx1, a ring nuclease (RN) and a target sequence that is complementary to the crRNA is transformed into E. …
Figure 4 with 1 supplement
Both faces of the Csx3 dimer are required for ring nuclease activity.
(A) Structure of the Csx3 dimer (monomers in cyan and green) bound to an RNA tetraloop (magenta) (PDB 3WZI), with residues D69 and H60 indicated. (B) Phosphorimage of TLC visualising cA4 (~200 nM) …
Figure 4—figure supplement 1
Metal dependence of Csx3.
Phosphor image of TLC visualising cA4 (~200 nM) cleavage by Csx3 (8 µM protein dimer) at 70°C when supplemented with 3 mM MnCl2, MgCl2, CaCl2 or CoCl2. Csx3 ring nuclease activity requires the …
Figure 5 with 5 supplements
Structure of the H60A variant of Csx3 in complex with cA4.
(A) Crystal structure of H60A Csx3 in complex with cA4. The crystal lattice reveals an extended filament with dimers of Csx3 sandwiching cA4. Three dimers are shown (green and cyan), with the cA4 in …
Figure 5—figure supplement 1
The crystal lattice of the Csx3:cA4 complex.
(A and B) Different views of the crystal lattice of Csx3 (mauve) in complex with cA4 (yellow). The asymmetric unit contains 5 dimers of Csx3, which with the crystallographic symmetry forms longer …
Figure 5—figure supplement 2
Schematic showing interactions between Csx3 and cA4.
The residues in blue and pink represent the two monomers in the dimer constituting the ‘D69 face’, and the residues in green and red represent the two monomers in the dimer constituting the ‘H60’ …
Figure 5—figure supplement 3
Superimposition of Csx3 in complex with cA4 and apo Csx3.
The active site of Csx3 (cyan and green, with the different colours representing different monomers in the dimer) in complex with cA4 (yellow) is superimposed with apo Csx3 (blue) (PDB: 3WZG). The …
Figure 5—figure supplement 4
Histidine residues in the active site of Csx3.
H57, H60 (H60 was not present in our structure, the position has been inferred by superposition with PDB 3WZI; both the alanine from our structure and superimposed histidine are shown for clarity), …
Figure 5—figure supplement 5
Investigation of H80A and R71A variants of Csx3.
TLC comparing ring nuclease activity of wild-type, H80A and R71A variants of Csx3 at 70°C. Both variants had severely reduced activity. Time points were 10, 60 and 600 s. Control c is reaction in …
Figure 6
Sigmoidal response of ring nuclease activity as a function of Csx3 concentration.
Plot visualising initial reaction rates of cA4 cleavage across increasing Csx3 concentrations. The cA4 concentration was 129 µM (25-fold greater than the highest concentration of protein assayed) …
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Figure 6—source data 1
Excel spreadsheet with raw data.
- https://cdn.elifesciences.org/articles/57627/elife-57627-fig6-data1-v2.xlsx
Figure 7
The ring nucleases.
The Crn1 family (represented here by Sso1393, PDB code 3QYF) is restricted to the crenarchaea (Athukoralage et al., 2018). The Csx1 family (represented here by PDB code 6O6Y) is self-limiting cA4-dep…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Sulfolobus solfataricus) | SsoCsm complex (eight subunits) | PMID:24119402 | virus expression construct | |
| Gene (Archaeoglobus fulgidus) | Csx3/Crn3 | PMID:26106927 | UniProtKB – O28415 | plasmid expression construct |
| Gene (Methanosarcina mazei) | MmCsx3 | This paper | UniProtKB - A0A0F8HGG9 | plasmid expression construct |
| Gene (Mycobacterium tuberculosis) | MtbCsm complex (five subunits) | PMID:31392987 | plasmid expression construct | |
| Gene (Thioalkalivibrio sulfidiphilus) | TsuCsx1 | PMID:31392987 | UniProtKB – B8GSI1 | plasmid expression construct |
| Gene (Thermoanaerobacterium phage THSA-485A) | AcrIII-1 | PMID:31942067 | UniProtKB - I3VYU1 | plasmid expression construct |
Additional files
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Supplementary file 1
Data collection and refinement statistics for H60A mutant of Csx3 in complex with cA4.
- https://cdn.elifesciences.org/articles/57627/elife-57627-supp1-v2.docx
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Supplementary file 2
Dynamic light scattering studies with AfCsx3.
- https://cdn.elifesciences.org/articles/57627/elife-57627-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57627/elife-57627-transrepform-v2.docx
