Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity
Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity
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DOI：
10.1016/j.molcel.2018.11.007
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发表时间：
2019-01-17
期刊：
影响因子：
16
通讯作者：
Patel, Dinshaw J.
中科院分区：
文献类型：
--
作者：
Jia, Ning;Mo, Charlie Y.;Patel, Dinshaw J.
III型CRISPR-Cas系统通过序列特异的RNase和靶RNA激活的序列非特异的DNase和RNase活性来提供对外来RNA和DNA的强大免疫力。我们报道了金尿酸热球菌CSM(CrRNA)二元、CSM(CrRNA)-靶RNA和CSM(CrRNA)-靶RNA(抗Tag)三元络合物在3.1埃范围内的冷冻-EM结构。CrRNA 5‘-Repeat标签的拓扑特征解释了定义靶切割位点的5’-标尺机制，5‘-Repeat内-2到-5位置的可及性作为避免自身免疫的传感器。Csm3拇指元件在crRNA-靶RNA双链中引入周期性扭曲，促进靶RNA以6-nt周期性切割。CSM(CrRNA)Csm1环段中的关键Glu残基采用一种建议的自我抑制构象，提示DNase活性调节。这些结构发现，加上对关键分子间接触的突变研究，为深入了解CSM(CrRNA)复合体组装、RNA靶向和位点特异性周期性切割的机制、DNA酶切割活性的调节和自身免疫抑制提供了见解。
Type III 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 Csm(crRNA) binary, Csm(crRNA)-target RNA and Csm(crRNA)-target RNA(anti-tag) ternary complexes in the 3.1 angstrom 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 Csm(crRNA) adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into Csm(crRNA) complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.