Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
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DOI：
10.1261/rna.070417.119
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发表时间：
2019-08-01
期刊：
影响因子：
4.5
通讯作者：
Dunkle, Jack A.
中科院分区：
文献类型：
--
作者：
Nasef, Mohamed;Muffly, Mary C.;Dunkle, Jack A.
CRISPR-Cas系统是原核生物中的一类适应性免疫系统，其使用小CRISPR RNA（crRNA）结合CRISPR相关（Cas）核酸酶来识别和降解外源核酸。最近的研究表明，III型CRISPR-Cas系统合成了先前未知存在于原核生物中的第二信使分子，环状寡腺苷酸（cOA）。这些分子激活Csm 6核酸酶以促进RNA降解，并且还可以协调对外源核酸的额外细胞应答。虽然cOA的产生已经被重建并表征为一些细菌和古细菌III型系统，但cOA的产生及其调节尚未被探索为表皮葡萄球菌III-A型CRISPR-Cas系统，这是CRISPR-Cas功能的长期模型。在这里，我们证明了该系统进行3-6 nt cOA的Mg 2+依赖性合成。我们表明，激活cOA合成的干扰之间的单核苷酸错配的crRNA和靶RNA在离散的位置，合成的拮抗Csm 3介导的靶RNA切割。总而言之，我们的研究结果确立了在III型CRISPR-Cas系统模型中产生cOA的要求，并提出了一旦外源RNA被破坏就会抑制免疫力的自然机制。
CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.