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, 45 (3), 303-13

Structure and Mechanism of the CMR Complex for CRISPR-mediated Antiviral Immunity

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Structure and Mechanism of the CMR Complex for CRISPR-mediated Antiviral Immunity

Jing Zhang et al. Mol Cell.

Abstract

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.

Figures

Figure 1
Figure 1. Purification of the CMR complex of S. solfataricus
A. UV trace showing fractions eluting from final MonoQ column, with CMR and RNAP complexes resolved. B. SDS-PAGE analysis of fractions from MonoQ column, showing separation of RNAP (fraction 30) from the 7-subunit CMR complex. C. Denaturing gel electrophoresis of end-labeled nucleic acid reveals the presence of RNA co-purifying with the CMR complex. The size range centered on 46 nt corresponds to a spacer with an 8 nt repeat-derived 5′-tag. D. Comparison of native and tagged versions of the CMR complex purified from S. solfataricus. Both tagged and untagged versions of Cmr7 are apparent, reflecting its higher stoichiometry in the complex. E. Mapping of Cmr1-7 onto the gene locus sso1986 to sso1992.
Figure 2
Figure 2. Distribution of crRNA bound by the S. solfataricus CMR complex
Examples of crRNA’s from all six CRISPR loci were observed, with a clear bias towards locus D, followed by locus C. The individual plots for each locus show that crRNA representation was highly variable, with adjacent spacers represented at levels that often varied by several orders of magnitude. For each graph, the X-axis represents the position of each spacer in the locus and the Y-axis represents the number of sequenced matches to each spacer. 88% of the total sequence reads were derived from locus D, which represents a nearly four-fold over-representation compared to the proportion of crRNAs encoded by that locus. CRISPR loci E and F, which are poorly transcribed, were significantly under-represented, as expected. However, crRNAs from CRISPRs A and B, which are highly transcribed and thought to be actively adding spacers in vivo, are also significantly under-represented in the CMR complex. The table shows the properties and representation in CMR of each CRISPR locus.
Figure 3
Figure 3. Crystal structures of two members of the Cmr7 family, viewed from the concave face of the dimer
A. The Cmr7 proteins Sso1986 (left) and Sso1725 (right) contain a structurally conserved core (yellow) and a variable region (blue and cyan for Sso1986 and Sso1725 respectively). The β13-β14 loop of Sso1986 is disordered and is represented as a dashed, black line. The N- and C-termini are represented as blue and red spheres respectively. B. Sso1986 and Sso1725 both form dimers and the structurally conserved core is located at the dimer interface. The interface itself is also conserved between the two proteins. C. The structurally conserved residues (green) and secondary structure (yellow) are located close to the dimer interface with a significant proportion positioned at the concave face. D. Electrostatic surface images show that the region of the concave face proximal to the dimer interface in both proteins (black box) have broad similarities.
Figure 4
Figure 4. 3D EM visualization of CMR complex
A. Surface representation of the Cmr2/3/7 sub-complex devoid of crRNA. B. Surface representation of the full CMR complex with bound crRNA. C. Superposition of Cmr2/3/7(blue surface) on CMR/RNA (black mesh). Black arrowheads point to regions of additional density on the full CMR complex with bound crRNA compared to Cmr2/3/7. Grey arrows indicate dimensions in Angstroms.
Figure 5
Figure 5. Characterization of the S. solfataricus CMR complex activity in vitro
A. Radiolabeled target RNA (5 μM) corresponding to spacer A1 was incubated with the Cmr7-tagged SsoCMR complex (0.5 μM) and guide RNA (1 μM). Cleavage of the target RNA was observed at six sites (labeled 1-6) (a). 3′ truncation of the guide RNA did not change the cleavage pattern of the target RNA, ruling out a molecular ruler mechanism (b). The 8 nt 5′-tag was essential for cleavage activity, as either deletion (c) or replacement with an 8A sequence (d) abolished nuclease activity. B. The D63 target (0.5 μM), was cleaved in the presence of Cmr7-tagged SsoCMR complex (0.5 μM), cognate guide RNA (0.1 μM) (e). Deletion of the unpaired 3′ end of the target RNA, which corresponds to the position of the PAM sequence, abolished activity (f). The presence of an unpaired 6A sequence at this position restored activity (g). C. Radiolabeled guide RNA corresponding to spacer A1 (3 μM) was incubated with the Cmr7-tagged SsoCMR complex (0.5 μM) and cognate target RNA (1 μM). Cleavage was observed at up to five positions, labeled 2′ to 6′ (h). This activity was dependent on the presence of the 3′ unpaired spacer sequence (i) and was not influenced by the presence of an additional unpaired extension at the 3′ end of the guide RNA (j, k). For each figure part, the 5′ end labeled RNA strand is indicated with an asterisk. Labeled Decade RNA markers (Ambion) are shown.
Figure 6
Figure 6. The S. solfataricus CMR complex cleaves RNA selectively at UA sites
A. Sequence map of the D63 target and guide RNA. B. A D63 target oligonucleotide (D631UA, 0.5 μM) with UA cleavage sites 1, 3, 4 and 5 mutated to UG, was labeled and incubated with the Cmr3-tagged CMR complex (0.5 μM) and a cognate guide RNA (crD631UA, 0.05 μM). Cleavage of the target RNA was only observed at the single remaining UA site, site 2. The same target paired with the wild-type guide RNA gave identical cleavage products despite the presence of 3 mismatches. C. A D63 guide RNA sequence with all four UA sites mutated to CA (crD630UA, 0.5 μM) was labeled and incubated with the Cmr3-tagged CMR complex (0.5 μM) and a target RNA containing a single UA site at position 2 (D631UA, 0.05 μM). No significant cleavage of this target sequence was observed. The standard guide RNA (crD63) was cleaved at all four UA sites under the same conditions, despite the presence of mismatches at 3 positions in the RNA duplex. Control lanes for all gels are: m1, Ambion Decade markers; c1, labeled RNA alone; c2, labeled RNA and CMR; c3, both RNA strands without CMR. Asterisks indicate the 5′ RNA end labeled by 32-P.

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