Control of cyclic oligoadenylate synthesis in a type III CRISPR system.
Control of cyclic oligoadenylate synthesis in a type III CRISPR system.
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DOI：
10.7554/elife.36734
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发表时间：
2018-07-02
期刊：
影响因子：
7.7
通讯作者：
White MF
中科院分区：
文献类型：
--
作者：
Rouillon C;Athukoralage JS;Graham S;Grüschow S;White MF
用于原核生物适应性免疫的CRISPR系统提供rna介导的病毒和移动遗传元件保护。当检测到病毒RNA转录物时，III型系统采用激活状态，允许DNA干扰和环低聚腺苷酸（cOA）的合成。辅酶a激活核酸酶和转录因子，协调抗病毒反应。我们证明，cOA的合成受到严格的时间控制，从目标RNA结合开始，并随着目标RNA被切割和解离而迅速失活。靶RNA中的错配是耐受良好的，并且仍然激活环化酶结构域，除非位于靶标的3 '端附近。硫代磷酸酯修饰减少靶RNA的切割并刺激cOA的产生。因此，最初归因于III型系统的“RNA切碎”活性可能反映了控制Cas10亚基的精细机制，而不是直接的抗病毒防御。近年来，通常简称为CRISPR的基因编辑工具已经广为人知。它的潜在应用范围很广，包括在研究、医疗保健和农业方面的应用。然而，CRISPR系统起源于微生物，它有助于保护微生物免受病毒感染。病毒通过将自己的基因插入宿主细胞而感染，CRISPR系统几乎像一把剪刀一样，可以切断病毒的DNA以阻止感染。CRISPR专家知道流行的CRISPR形式是II型，但还有其他类型。III型CRISPR作为一种遗传工具用处不大，但也可以保护微生物免受病毒的侵害。除了靶向DNA外，III型CRISPR还靶向来自病毒的相关RNA分子。当它遇到来自病毒的RNA时，III型CRISPR会产生一种叫做环寡腺苷酸（简称cOA）的小分子。辅酶a分子可以激活一种叫做非特异性核糖核酸酶的酶，这种酶可以破坏细胞中的所有RNA。与II型CRISPR相比，这种防御没有那么微妙，而且还可以通过破坏微生物赖以生存的其他RNA分子来破坏细胞。因此，适当的调控是必不可少的，以防止III型系统不必要地杀死感染细胞。Rouillon等人研究了火山泉中的嗜热微生物Sulfolobus solfataricus对III型CRISPR系统的控制。这个物种多年来一直是CRISPR系统研究的一个模型，部分原因是它的蛋白质非常稳定，这使得它们更容易在实验室中使用。结果表明，III型CRISPR通过将四分子三磷酸腺苷（ATP）结合成一个环来制造cOA。CRISPR会立即对细胞中的病毒RNA做出反应。一旦RNA开始被破坏，它也会与RNA分离。产生cOA的快速激活和沉默确保了CRISPR系统受到严格控制。这些发现表明，cOA的产生与病毒RNA的丰度密切相关，确保了对感染的比例和及时反应。使用cOA可以增强细胞的反应，因为它允许单个RNA分子激活更大的变化。III型CRISPR系统在自然界广泛存在，更好地了解它们可以提高酸奶等依赖于健康细菌的产品的产量；目前，病毒给这个行业造成了很大的经济损失。该领域的进一步研究也可能导致过度激活III型CRISPR来破坏细菌细胞的新抗生素。
The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3’ end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The ‘RNA shredding’ activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence. The gene editing tool often known simply as CRISPR has become well known in recent years. Its potential applications are wide ranging, including uses in research, healthcare and agriculture. Yet, the CRISPR system originated in microbes where it helps to protect them from viral infections. Viruses infect by inserting their own genes into a host cell, and – almost like a pair of scissors – the CRISPR system can cut up the virus’s DNA to stop infections. CRISPR experts know the popular form of CRISPR as type II, but there are others. Type III CRISPR is less useful as a genetic tool but does also protect microbes from viruses. In addition to targeting DNA, type III CRISPR targets the related RNA molecules from viruses. When it encounters RNA from a virus, the type III CRISPR produces a small molecule called cyclic oligoadenylate (or cOA for short). The cOA molecule activates enzymes known as non-specific ribonucleases, which can destroy all the RNA in the cell. This defence is a less subtle than that provided type II CRISPR and can also damage the cell by destroying other RNA molecules that the microbes use to survive. As such, proper regulation is essential to prevent the type III system from unnecessarily killing the infected cell. Rouillon et al. studied the control of the type III CRISPR system from the heat-loving microbe Sulfolobus solfataricus, which is found in volcanic springs. This species has been a model for studies of the CRISPR system for many years, in part because its proteins are very stable which makes them easier to work with in the laboratory. The results show that the type III CRISPR makes cOA by combining four molecules of adenosine triphosphate (ATP) into a ring. CRISPR responds immediately to viral RNA in the cell. It also detaches from the RNA as soon as it starts to be destroyed. Rapid activation and silencing of the production cOA ensures that the CRISPR system is tightly controlled. These findings reveal that cOA production is tightly linked to the abundance of viral RNA, ensuring a proportional and timely response to infection. Using cOA amplifies the cell's response because it allows a single RNA molecule to activate a larger change. Type III CRISPR systems are widespread in nature, and a better understanding of them could improve the yield of products, like yoghurt, that depend on healthy bacteria; currently viruses cause a lot of economic damage in this industry. Further research in this area could also lead to new antibiotics that over-activate type III CRISPR to destroy bacterial cells.