Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
Biolog Life Science Institute GmbH & Co. KG, Flughafendamm 9a, D-28199, Bremen, Germany.
Nat Commun. 2020 Mar 27;11(1):1596. doi: 10.1038/s41467-020-15334-5.
Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.
细菌和古菌的 CRISPR-Cas 系统为抵御噬菌体和质粒等遗传入侵物提供了 RNA 导向的免疫。在靶 RNA 识别后，III 型 CRISPR-Cas 系统产生环状寡聚腺苷酸第二信使，通过其 CARF 结构域激活下游效应物，包括 Csm6 核糖核酸酶。在这里，我们表明肠球菌意大利亚种 Csm6（EiCsm6）降解其同源环状六聚 AMP（cA6）激活剂，并报告了与 cA6 模拟物结合的 EiCsm6 的晶体结构。我们的结构、生化和体内功能测定揭示了 CARF 结构域如何识别 cA6 以激活 Csm6 的 HEPN 结构域进行副产物 RNA 降解，以及 CARF 结构域介导的 cA6 切割如何为 Csm6 活性提供内在的关闭开关，从而在没有环核酶的情况下限制 Csm6 活性。这些机制有助于快速清除入侵物，并确保 CRISPR 干扰的终止以限制自我毒性。
