Molecular Cell
Volume 73, Issue 2, 17 January 2019, Pages 264-277.e5
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Article
Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity

https://doi.org/10.1016/j.molcel.2018.11.007Get rights and content
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Highlights

  • Ruler mechanism based on 5′-repeat tag dictates positioning of RNA cleavage sites

  • Kinks in crRNA-target RNA duplex identify periodic RNA cleavage at 6-nt intervals

  • Pairing potential with positions -2 to -5 within the 5′-repeat regulate autoimmunity

  • A Glu-rich loop adopts an autoinhibitory conformation regulating DNase activity

Summary

Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA-target RNA and CsmcrRNA-target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5′-repeat tag explains the 5′-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5′-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of CsmcrRNA adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into CsmcrRNA complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.

Keywords

cryo-EM
type III-A CRISPR-Cas complexes
T. onnurineus Csm
ternary complex assembly with crRNA and target RNA
5′ repeat tag pairing controls autoimmunity
RNA cleavage with 6-nt periodicity
DNase activity regulation by Glu residues

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