Characterization of CRISPR RNA Biogenesis and Cas6 Cleavage-Mediated Inhibition of a Provirus in the Haloarchaeon Haloferax mediterranei
Characterization of CRISPR RNA Biogenesis and Cas6 Cleavage-Mediated Inhibition of a Provirus in the Haloarchaeon Haloferax mediterranei
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地中海盐古菌 Haloferax mediterranei 中 CRISPR RNA 生物发生和 Cas6 切割介导的原病毒抑制的表征
DOI：
10.1128/jb.01688-12
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发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
--
作者：
Li, Ming;Liu, Hailong;Xiang, Hua
摘要由CRISPR(簇规则间隔短回文重复序列)阵列和Cas(CRISPR相关)基因组成的适应性免疫系统在广泛的细菌和古菌中被发现，最近引起了广泛的研究。然而，在海洋生物中，I-B亚型CRISPR-Cas系统的特征较少。在这里，我们研究了Cas6介导的RNA在Haloferax Mediterranei中的加工。实验确定了Cas6裂解位点和CRISPR转录起始位点，并检测到CRISPR转录本的加工从早期对数到静止阶段的递增模式。用遗传学的方法，我们发现Cas1、Cas3或Cas4的缺失意外地导致CRISPR转录本的减少，而Cas5、Cas6和Cas7被发现在稳定成熟的CRISPR RNA(CrRNA)方面是必不可少的。有趣的是，我们观察到CRISPR和Cas3独立于CRISPR和Cas3对缺陷前病毒的抑制，其中假定的Cascade(CRISPR相关的抗病毒防御复合体)蛋白(Cas5、Cas6、Cas7和Cas8b)是必不可少的。前病毒转录本携带的一个序列被发现与CRISPR重复RNA同源，容易受到Cas6介导的切割，这意味着一种独特的干扰机制可能解释了这种不寻常的抑制。这些结果为嗜盐古细菌中的I-B亚型CRISPR-Cas系统提供了基础信息，并提示了CRISPR-Cas系统的多种机制和多种生理功能。
ABSTRACT The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.