Programmable RNA Shredding by the Type III-A CRISPR-Cas System of Streptococcus thermophilus

被引:228
|
作者
Tamulaitis, Gintautas [1 ]
Kazlauskiene, Migle [1 ]
Manakova, Elena [1 ]
Venclovas, Ceslovas [2 ]
Nwokeoji, Alison O. [3 ]
Dickman, Mark J. [3 ]
Horvath, Philippe [4 ]
Siksnys, Virginijus [1 ]
机构
[1] Vilnius State Univ, Inst Biotechnol, Dept Prot DNA Interact, LT-02241 Vilnius, Lithuania
[2] Vilnius State Univ, Inst Biotechnol, Dept Bioinformat, LT-02241 Vilnius, Lithuania
[3] Univ Sheffield, ChELSI Inst, Dept Chem & Biol Engn, Sheffield S1 3JD, S Yorkshire, England
[4] DuPont Nutr & Hlth, F-86220 Dange St Romain, France
基金
英国工程与自然科学研究理事会; 英国生物技术与生命科学研究理事会;
关键词
ANTIVIRAL DEFENSE; ADAPTIVE IMMUNITY; COMPLEX; DNA; CASCADE; PROTEIN; MECHANISM; ENDONUCLEASE; PROKARYOTES; EVOLUTION;
D O I
10.1016/j.molcel.2014.09.027
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.
引用
收藏
页码:506 / 517
页数:12
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