A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator
A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator
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DOI：
10.1101/582114
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发表时间：
2019-03
影响因子：
5.6
通讯作者：
Januka S. Athukoralage;S. Graham;Sabine Grueschow;Christophe Rouillon;M. F. White
中科院分区：
文献类型：
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作者：
Januka S. Athukoralage;S. Graham;Sabine Grueschow;Christophe Rouillon;M. F. White
环寡腺苷酸（cOA）第二信使由III型CRISPR系统响应于病毒感染而产生。cOA变构激活CRISPR辅助核糖核酸酶Csx 1/Csm 6，其使用HEPN（高等真核生物和原核生物，核苷酸结合）活性位点非特异性降解RNA。这提供了有效的免疫力，但也可能导致受感染细胞的生长停滞，一旦病毒感染被清除，就需要一种使核糖核酸酶失活的方法。在泉古菌中，专用的环状核酸酶降解cA 4（cOA由4个AMP单位组成），但在细菌中尚未鉴定出等效的酶。我们证明，在嗜热栖热菌HB 8，未表征的蛋白TTHB 144是cA 4激活的HEPN核糖核酸酶，也降解其激活剂。TTHB 144在N-末端CARF（CRISPR相关罗斯曼折叠）结构域结合并降解cA 4。这两种活性可以通过定点诱变分离。TTHB 144是自限性CRISPR核糖核酸酶的第一个例子。图形摘要
Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity, but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterised protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR Associated Rossman Fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease. Graphical abstract