Crystal Structure of the Cmr2–Cmr3 Subcomplex in the CRISPR–Cas RNA Silencing Effector Complex

Osawa, Takuo; Inanaga, Hideko; Numata, T.

doi:10.1016/j.jmb.2013.03.042

Cited by 42 publications

(42 citation statements)

References 43 publications

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“…2; Spilman et al 2013). Another recent study found that the Cmr2-Cmr3 complex could bind the crRNA and target RNA in the absence of the other Cmr proteins (Osawa et al 2013); however, we did not observe specific RNA binding by these two proteins under the conditions used in our work (with competitor RNA to reduce nonspecific interactions). Four Cmr4 subunits form a helical chain (Staals et al 2013; N Ramia, M Spilman, L Tang, Y Shao, J Elmore, CR Hale, A Cocozaki, N Bhattacharya, RM Terns, Figure 7.…”

Section: Crrna Binding Requires Four Cmr Proteinscontrasting

confidence: 65%

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Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

et al. 2014

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The effector complex of the Cmr/type III-B CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) system cleaves RNAs recognized by the CRISPR RNA (crRNA) of the complex and includes six protein subunits of unknown functions. Using reconstituted Pyrococcus furiosus Cmr complexes, we found that each of the six Cmr proteins plays a critical role in either crRNA interaction or target RNA capture. Cmr2, Cmr3, Cmr4, and Cmr5 are all required for formation of a crRNA-containing complex detected by native gel electrophoresis, and the conserved 59 repeat sequence tag and 59-OH group of the crRNA are essential for the interaction. Interestingly, capture of the complementary target RNA additionally requires both Cmr1 and Cmr6. In detailed functional studies, we determined that P. furiosus Cmr complexes cleave target RNAs at 6-nucleotide (nt) intervals in the region of complementarity, beginning 5 nt downstream from the crRNA tag and continuing to within~14 nt of the 39 end of the crRNA. Our findings indicate that Cmr3 recognizes the signature crRNA tag sequence (and depends on protein-protein interactions with Cmr2, Cmr4, and Cmr5), each Cmr4 subunit mediates a target RNA cleavage, and Cmr1 and Cmr6 mediate an essential interaction between the 39 region of the crRNA and the target RNA.

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Section: Crrna Binding Requires Four Cmr Proteinscontrasting

confidence: 65%

“…1, 3). Cmr2 interacts with Cmr3 (Osawa et al 2013;Shao et al 2013;Spilman et al 2013;Staals et al 2013) and with the guide sequence of the crRNA nearest the 59 tag ( Fig. 2; Spilman et al 2013).…”

Section: Crrna Binding Requires Four Cmr Proteinsmentioning

confidence: 99%

Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

et al. 2014

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“…Crystal Structure Determination of P. furiosus Cmr Proteins Previous structural studies have revealed the crystal structures of a core fragment of Pf Cmr2 either in isolation (Cocozaki et al, 2012;Zhu and Ye, 2012) or in complex with Pf Cmr3 (Osawa et al, 2013;Shao et al, 2013), as well the crystal structure of Pf Cmr5 (Park et al, 2013). We determined the atomic models of the remaining subunits and domains of the Pf Cmr complex.…”

Section: Resultsmentioning

confidence: 99%

“…Studies to date have revealed the crystal structures of three of the six Pf Cmr subunits (Cmr2, Cmr3, and Cmr5; Cocozaki et al, 2012;Osawa et al, 2013;Park et al, 2013;Shao et al, 2013;Zhu and Ye, 2012; reviewed in Reeks et al, 2013) and the overall superhelical architecture of the complex by cryoelectron microscopy (cryo-EM) (Spilman et al, 2013). The Pf Cmr complex can load either a 39 nt long or a 45 nt long crRNA .…”

Section: Introductionmentioning

confidence: 99%

Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4

et al. 2014

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The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.

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“…11,17 In recent years, structural information on Cas proteins has started to provide insights into the molecular mechanisms of crRNA binding and target recognition. The combination of X-ray crystallography 8,[18][19][20][21][22] and electron microscopic studies of the type I Cascade [23][24][25] and of the Type III-B Cmr-complex [26][27][28] has shown how some of the Cas proteins interact and bind crRNA.…”

Section: Introductionmentioning

confidence: 99%

Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

Hrle

Ebert

et al. 2013

RNA Biology

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The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 angstrom resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins

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Crystal Structure of the Cmr2–Cmr3 Subcomplex in the CRISPR–Cas RNA Silencing Effector Complex

Cited by 42 publications

References 43 publications

Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4

Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

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