Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog

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

doi:10.1016/j.molcel.2015.03.018

Cited by 133 publications

(189 citation statements)

References 53 publications

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Section: Type I and Type Iii Crispr Systemsmentioning

confidence: 99%

“…However, in case of type III systems, pre-CRISPR transcript is processed by Cas6 in solitude and not as part of the effector complex (Carte et al, 2008; Carte et al, 2010;Wang et al, 2011;Shao and Li, 2013;Shao et al, 2016; Makarova et al, 2017a). Further, Cas6/III does not become a constituent of interference complex (Hale et al, 2009;Zhang et al, 2012;Rouillon et al, 2013;Spilman et al, 2013; Benda et al, 2014;Staals et al, 2014;Osawa et al, 2015;Taylor et al, 2015; Makarova et al., 2017a), with the exception being Csm/III-A complex from Streptococcus thermophilus that shows weak transient interactions with Cas6 (Tamulaitis et al, 2014). This possibly grants Cas6/ III the flexibility required to associate with multiple subtypes that potentially differ at the interference stage.…”

mentioning

confidence: 99%

“…This allows sharing of crRNA processing pathways, with the mature crRNAs ultimately getting loaded onto specific effector complex. Type III system displays two kinds of effector complexes -Csm (Type III-A/D) or Cmr (Type III-B/C) (Hale et al, 2009;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Tamulaitis et al, 2014;Zhang et al, 2016), which show distant relationship with Type I effector complex (Rouillon et al, 2013;Osawa et al, 2015;Taylor et al, 2015).A primary processed crRNA typically contains 8 nt repeat sequence at the 5'-end (often called 5' handle), a spacer sequence (guide) (Brouns et al, 2008; Haurwitz et al, 2010; Lintner et al, 2011) and a variable 3'-end in some instances, which stems from further trimming at 3'-end by as yet unidentified nuclease(s) (Hale et al, 2008; Hatoum-Aslan et al, 2011;Richter et al, 2012;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Shao and Li, 2013;Tamulaitis et al, 2014). Intriguingly, the extent of processing appears to be effector complexspecific (Hale et al, 2009; Lintner et al, 2011;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Tamulaitis et al, 2014).…”

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Cutting it Right: Plasticity and Strategy of CRISPR RNA Specific Nucleases

Punetha

Yoganand

Nimkar

et al. 2017

Proceedings of the Indian National Science Academy

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The existence of adaptive immunity in prokaryotes came to light with the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in association with CRISPR-associated (Cas) proteins. This RNA mediated defence system confers resistance against the invading mobile genetic elements such as phages and plasmids. The CRISPR-Cas system operates by forming a ribonucleoprotein complex that comprises of an invader derived small RNA and Cas protein(s). Herein the small RNA acts as a guide to recognize the nucleic acid target whereas the Cas proteins facilitate target annihilation. Given the cardinal role adopted by this small RNA, its maturation from the pre-CRISPR transcript forms a pivot for successful adaptive immunity. The mandate to generate the guide CRISPR RNA (crRNA) is fulfilled by specific endoribonuclease, which processes the pre-crRNA transcript in between the repeats to liberate the individual interfering units. Intriguingly, while some endoRNases of the CRISPR system are able to process the pre-crRNA independently, others require participation of additional Cas proteins, which form a multi-protein complex for processing the pre-crRNA. Additionally, some CRISPR variants require non-Cas auxiliary factors to process the pre-crRNA. The mode of crRNA maturation further diversifies as the endoRNases in CRISPR variants coevolve with repeat clusters that exhibit high diversity in sequence and folding. Therefore, the maturation of a specific crRNA requires a distinct mechanistic solution for substrate discrimination by these endoRNases, the understanding of which is essential for appreciating the CRISPR biology. This review highlights the vivid modes adopted by the diverse CRISPR-Cas systems to generate the mature crRNA. Keywords: CRISPR RNA; CRISPR-Cas IntroductionIn order to survive, all organisms must overcome their predators. The prokaryotes and their viral predators coexist in natural and man-made environment and therefore the prokaryotes face a constant threat of getting infected by phages. This results in acute pressure on the microbial community to coevolve with their predators causing an evolutionary arms race between prey and predator. Pitted against a hostile environment, prokaryotes have developed multilayered antiviral defense systems, which act at various stages of the infection cycle of the invader. These include various innate defense systems like surface exclusion (receptor downregulation or masking), super infection exclusion (Sie systems), restrictionmodification systems (R-M and R-M like systems), and abortive infection systems (Abi) (Hyman and Abedon, 2010; Labrie et al., 2010;Westra et al., 2012a). These innate defense mechanisms are diffusive in nature and do not rely on the identity of the predator to elicit a response (Fig 1). Added to this repertoire of arsenals, the recently discovered Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in association with CRISPRassociated (Cas) proteins endows the bacteria and archaea with an adaptive immunity (Ja...

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Section: Type I and Type Iii Crispr Systemsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Cutting it Right: Plasticity and Strategy of CRISPR RNA Specific Nucleases

Punetha

Yoganand

Nimkar

et al. 2017

Proceedings of the Indian National Science Academy

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“…Electron microscopy data and protein crystallographic studies have revealed in detail the composition and function of the Cascade [22][23][24][25][26] and the Cmr crRNP complexes [17,27]. Furthermore, electron microscopy and MS data have defined the Csm crRNP complex from Sulfolobus solfataricus, which is formed by up to seven different proteins [28] and a homologous complex in Thermus thermophilus that consists of five different proteins: Csm1-5 [18].…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, electron microscopy and MS data have defined the Csm crRNP complex from Sulfolobus solfataricus, which is formed by up to seven different proteins [28] and a homologous complex in Thermus thermophilus that consists of five different proteins: Csm1-5 [18]. Cmr, Csm and Cascade complexes show overall structural similarity because they are formed by structurally and functionally related subunits [27,28]. In the present study, aiming to obtain more more information about the Csm crRNP complex, we cloned, expressed, purified, crystallized and solved the structure of the Csm2 protein of Thermotoga maritima MSB8.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for dimer formation of the CRISPR‐associated protein Csm2 of Thermotoga maritima

et al. 2016

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The clusters of regularly interspaced short palindromic repeats (CRISPR) and the Cas (CRISPR‐associated) proteins form an adaptive immune system in bacteria and archaea that evolved as an RNA‐guided interference mechanism to target and degrade foreign genetic elements. In the so‐called type IIIA CRISPR‐Cas systems, Cas proteins from the Csm family form a complex of RNPs that are involved in surveillance and targeting tasks. In the present study, we report the crystal structure of Thermotoga maritima Csm2. This protein is considered to assemble into the helically shaped Csm RNP complex in a site opposite to the CRISPR RNA binding backbone. Csm2 was solved via cadmium single wavelength anomalous diffraction phasing at 2.4 Å resolution. The structure reveals that Csm2 is composed of a large 42 amino‐acid long α‐helix flanked by three shorter α‐helices. The structure also shows that the protein is capable of forming dimers mainly via an extensive contact surface conferred by its long α‐helix. This interaction is further stabilized by the N‐terminal helix, which is inserted into the C‐terminal helical portion of the adjacent subunit. The dimerization of Csm2 was additionally confirmed by size exclusion chromatography of the pure recombinant protein followed by MS analysis of the eluted fractions. Because of its role in the assembly and functioning of the Csm CRISPR RNP complex, the crystal structure of Csm2 is of great importance for clarifying the mechanism of action of the subtype IIIA CRISPR‐Cas system, as well as the similarities and diversities between the different CRISPR‐Cas system. Database The structure of Thermotoga maritima Csm2 has been deposited in the Protein Data Bank under accession code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5AN6.

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Structure of Csm2 elucidates the relationship between small subunits of CRISPR‐Cas effector complexes

Venclovas

2016

FEBS Letters

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Type I and type III CRISPR-Cas effector complexes share similar architecture and have homologous key subunits. However, the relationship between the so-called small subunits of these complexes remains a contentious issue. Here, it is shown that the recently solved structure of Thermotoga maritima Csm2 represents a dimer with the extensive structure swapping between monomers. Unswapping the structure generates a compact globular monomer which shares similar structure and surface properties with Cmr5, the small subunit of a related Cmr complex. Detailed analysis of available structures of small subunits reveals that they all have a common fold suggesting their common origin.

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Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog

Cited by 133 publications

References 53 publications

Cutting it Right: Plasticity and Strategy of CRISPR RNA Specific Nucleases

Cutting it Right: Plasticity and Strategy of CRISPR RNA Specific Nucleases

Structural basis for dimer formation of the CRISPR‐associated protein Csm2 of Thermotoga maritima

Structure of Csm2 elucidates the relationship between small subunits of CRISPR‐Cas effector complexes

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