Structure of an RNA silencing complex of the CRISPR-Cas immune system - PubMed (original) (raw)

Comparative Study

Structure of an RNA silencing complex of the CRISPR-Cas immune system

Michael Spilman et al. Mol Cell. 2013.

Abstract

Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs (crRNAs) containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multicomponent Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here, we report cryoelectron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ~12 Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits: Cmr2 and Cmr3 at the conserved 5' crRNA tag sequence and Cmr1 and Cmr6 near the 3' end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Fig. 1

Fig. 1

Overview of the P. furiosus CRISPR/Cas locus and the Cmr complex structure determined by cryo electron microscopy. (A) The genes encoding the Cmr complex protein subunits are color-coded to match those used for structure images in subsequent figures. The CRISPR repeats are shown in black and spacers in various colors. (B) The mature 39-nt and 45-nt crRNAs contain a 5′ repeat-derived 8-nt sequence (5′-tag) and a 31-nt or 37-nt spacer-derived sequence (guide), respectively. The sequence of the target RNA used in RNA cleavage assays and assembly with the Cmr complex is shown in blue. (C) Color-coded EM density of the Cmr complex bound with the 45-nt crRNA and the target RNA is shown in two orientations. Cmr1 (red), Cmr2 (light blue), Cmr3 (orange), Cmr4 (three different shades of green), Cmr5 (three different shades of yellow), and Cmr6 (magenta). (D) Fitting of the crystal structure of Cmr2-Cmr3 complex to the ‘foot’ of the Cmr complex in two orthogonal orientations. (E) Helical reconstruction of the Cmr4-Cmr5 filament structure. Circled regions indicate matched densities in both the Cmr complex and the Cmr4-Cmr5 filament. See also Figures S1, S2 and S3.

Fig. 2

Fig. 2

Assembly process of the Cmr complex. (A) Left, a ribbon model of the assembled protein subunits of an intact target-bound Cmr complex. Right, capping at the foot of the Cmr complex by interaction of the homologous domain of Cmr2 with Cmr5 and of Cmr3 with Cmr4. Structural similarity between Cmr5 ((Park et al., 2013), PDB ID 4GKF) and the D4 domain of Cmr2 is shown (r.m.s.d = 3.3 Å for 62 aligned Cα atoms). A homology model of Cmr4 constructed from Cas6 (sequence similarity 30%, (Carte et al., 2008), PDB ID 3PKM) is also homologous to that of Cmr3 (r.m.s.d = 3.4 Å for 136 aligned Cα atoms). (B) A micrograph of the Cmr4-Cmr5 filament and the class averages of Cmr4-Cmr5 filaments. (C) Model for assembly method of the Cmr complex. By acting as a non-extendable Cmr4-Cmr5 dimer, Cmr6 and Cmr2-Cmr3 complex cap the growth of the Cmr4-Cmr5 filament at the top and foot of the Cmr complex, respectively. See also Figures S2E and S4.

Fig. 3

Fig. 3

Positions of the crRNA and target RNA within the complex. (A) RNA Substrates used for crosslinking assays. The 45-nt crRNAs (1-3, 5-8) or tag RNAs (4) were generated by in vitro transcription and subsequent Cas6 digestion. Regions marked in yellow contain radiolabeled nucleotides. Labeled (i.e. crosslinked) (B) and Coomassie-stained (i.e. total) (C) proteins are shown. The RNA from (A) used in each reaction is indicated beneath the lanes, and correlates with (A). T1 and A denote RNases used in the experiment and visible in the Coomassie-stained gel (C). (D) Difference density to identify bound target RNA. The difference density was obtained by subtracting the negatively stained density of the Cmr complex in the absence of target RNA from that in the presence of target RNA. Left, the common density +/- target RNA is shown in yellow and the difference density in blue (10σ). Right, the same view as that on the left but clipped to expose the difference density in the center of the complex. (E) Schematic structure of the holo Cmr complex showing the deduced path of the crRNA and target RNA. See also Table S1.

Fig. 4

Fig. 4

Conserved organization of an RNA- and DNA-targeting CRISPR-Cas effector complex. The Cse and Cmr CRISPR-Cas systems include distantly related members of several broad categories of Cas proteins: large subunit (blue), small subunit (shades of yellow), Cas5 superfamily (orange), Cas7 superfamily (shades of green) and Cas6 superfamily proteins (gray). Our findings reveal a conserved functional organization of these classes of Cas proteins within the effector complexes. (A). Comparison of cryoEM structures of E. coli Cascade (left) and P. furiosus Cmr complex (right). Segmented densities assigned to the subunits are color-coded according to their broad classification. (B). Cartoon representations of the functional organization of the complexes. The Cas5 superfamily RAMP proteins – Cmr3 and Cas5e – are found near the 5′ tag of the crRNAs in both complexes. Large subunit proteins – Cmr2 (member of the Cas10 superfamily) and Cse1 (member of the Cas8 superfamily) – are also found near the 5′ end of the crRNAs. Cas7 superfamily RAMP proteins – Cmr4, Cmr6 and Cmr1 in the Cmr complex and Cse4 in the Cse complex – and the small subunit of proteins – Cmr5 and Cse2 – form the backbones of the helical structure that extends along the guide region of the crRNAs. In Cascade, the Cas6 superfamily RAMP protein – Cse3 – remains associated with the CRISPR repeat sequence retained at the 3′ end of the crRNA. The Cse complex binds DNA targets that are then cleaved by Cas3. The Cmr complex cleaves complementary RNAs.

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