The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.
The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.
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DOI：
10.1261/rna.039842.113
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发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Terns MP
中科院分区：
文献类型：
--
作者：
Sheppard NF;Glover CV 3rd;Terns RM;Terns MP
原核生物经常暴露于来自质粒、质粒和转座子的潜在有害的侵入性核酸。一种防御方法是CRISPR-Cas适应性免疫系统。不同的CRISPR-Cas系统形成不同的核糖核蛋白效应子复合物，其靶向并切割侵入性核酸以提供免疫。已发现III-B型Cmr效应复合物在其已被表征的各种细菌和古细菌生物体中靶向入侵者的RNA和DNA。有趣的是，在许多基因组中，编码Csx 1蛋白的基因经常位于Cmr 1 -6基因附近，暗示Csx 1在Cmr功能中的作用。然而，有证据表明，Csx 1是不是一个稳定相关的CMR效应复合物的组成部分，但必要的CMR系统在Sulfolobus islandicus的DNA沉默。为了研究Csx 1蛋白的功能，我们表征了重组激烈火球菌Csx 1对各种核酸底物的活性。我们表明，Csx 1是一种金属不依赖性，核糖核酸内切酶，选择性地作用于单链RNA和特异性切割后的腺苷。Csx 1的RNA切割活性依赖于位于蛋白质C-末端结构域内的保守HEPN基序。该基序也是其他已知核糖核酸酶活性的关键。总的来说，这些发现表明，III-B型CRISPR-Cas系统的入侵者沉默依赖于来自Cmr效应复合物的RNA和DNA核酸酶活性以及附属的反式作用Csx 1内切核糖核酸酶。
Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.