A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems

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Science  11 Aug 2017:
Vol. 357, Issue 6351, pp. 605-609
DOI: 10.1126/science.aao0100
  • Fig. 1 StCsm complex–mediated conversion of ATP to the reaction products.

    (A) Schematic organization of the S. thermophilus type III-A CRISPR-Cas locus. Spacers interspaced between repeats are numbered. The transcribed CRISPR RNA is processed and bound by Cas proteins to form a S. thermophilus Csm (StCsm) complex. The mature crRNA guides the StCsm complex in the recognition of an invader transcript at the interference stage. (B) Target RNA sequence requirements for the StCsm-mediated conversion of ATP to the reaction products. Cartoons above the gels depict reaction components; the target sequence is blue, the complementary strand (matching spacer in crRNA) is red, and the 3′-flanking sequence of protospacer complementary to the 5′-handle of crRNA is pink. (C) Effect of mutations in StCsm Cas10 HD and Palm domains on the conversion of ATP to the reaction products. The domain architecture of the StCas10 protein is presented above the gels. HD denotes an HD-type phosphohydrolase/nuclease domain (blue); the two Palm domains are polymerase/cyclase–like Palm domains, one of which contains a GGDD motif (green); D2 and D4 denote α-helical domains (gray). Conserved active site residues subjected to alanine mutagenesis are indicated above the colored boxes. M, ATP partial thermal hydrolysis ladder; wt, wild type. Throughout, single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; G, Gly; H, His; K, Lys; N, Asn; Q, Gln; R, Arg; S, Ser; T, Thr; X, any amino acid.

  • Fig. 2 Identification of the StCsm-mediated ATP reaction products.

    (A) Treatment of ATP reaction products with T4 polynucleotide kinase and PDE12 or P1 nuclease. The tri-adenylate product (gray arrow) migrates differently from linear A3, indicative of its nonlinear structure. Pi, (pyro)phosphate; PNK, T4 polynucleotide kinase. (B) Characterization of ATP reaction products by electrospray ionization–MS analysis. Identified masses (Mwexp, in daltons) and compounds are presented above the HPLC chromatogram. A260, absorbance at 260 nm; AU, arbitrary units. (C) HPLC-MS analysis of the tri-adenylate digest by P1 nuclease (left) and scheme for P1-mediated cA3 or linear A3 hydrolysis (right). (D) Proposed model for the StCsm-mediated ATP polymerase/cyclase reaction mechanism. Both StCas10 Palm domains (P and P*) can bind ATP, but only one features the conserved GGDD motif and is catalytically active. ATP binding in the P site positions the 3′-OH for nucleophilic attack on α-phosphorous of ATP bound in the P* site to produce pppA2. pppA2 can then rearrange between P and P*, positioning the 3′-OH group for internal nucleophilic attack on its own triphosphate moiety to yield cA2 or translocate between the sites to react with a new ATP molecule and produce pppA3. Subsequent polymerization terminated by cyclization results in cA3 to cA6. (E) HPLC-MS analysis and reaction scheme of StCsm-mediated conversion of synthetic linear OA triphosphates into the corresponding cOAs. (M-H) and (M-2H)−2 denote ion mass/charge ratio values for different ionization forms. Mwtheor denotes the predicted molecular mass in daltons.

  • Fig. 3 Csm6 ribonuclease activation by StCsm-produced cOAs.

    (A) Domain arrangement of StCsm6. Conserved residues subjected to alanine mutagenesis are indicated above the colored boxes and as red spheres in the ribbon structures below. Blue mesh denotes the putative ligand binding cleft. (B) StCsm6-mediated ssRNA cleavage in the absence and presence of 0.5 nM cA6. Red indicates the StCsm6 concentration that is required for efficient ssRNA hydrolysis. (C) RNase activity of StCsm6 mutants. (D) Dependence of StCsm6 RNase activity on nucleotide effectors. (E) Dependence of TtCsm6 RNase activity on nucleotide effectors. Chemical structures of cA4 and cA6 are shown on the right.

  • Fig. 4 Mechanism of signaling in type III CRISPR-Cas systems.

    A transcript from an invading DNA serves as a stimulus for the StCsm complex (RNA Pol, RNA polymerase). Recognition of the invasive transcript by the Csm complex through base-pairing between the crRNA and the transcript promotes the three activities of Csm: (i) Csm3-mediated cleavage of the transcript itself, (ii) degradation of the corresponding invading DNA by the HD domain of Cas10, and (iii) synthesis of cOAs by the Palm domain of Cas10. The resultant cOA is a signaling molecule that is recognized by the sensory CARF domain in Csm6, which in turn activates the effector HEPN domain of Csm6. Thus, activated Csm6 effectively degrades ssRNA, which could buy the time necessary to ensure the destruction of the invasive genome or eventually lead to cell death.

Supplementary Materials

  • A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems

    Migle Kazlauskiene, Georgij Kostiuk, Česlovas Venclovas, Gintautas Tamulaitis, Virginijus Siksnys

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S24
    • Table S1
    • References

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