The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Haihe Laboratory of Cell Ecosystem, Tianjin Institute of Immunology, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
Nucleic Acids Res. 2022 Dec 9;50(22):12913-12923. doi: 10.1093/nar/gkac1151.
The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.
III-E 型 CRISPR-Cas 系统是原核生物中新发现的适应性免疫系统，它使用单个 Cas7-11 蛋白特异性切割靶 RNA。Cas7-11 可以与 Csx29 结合，Csx29 是一种假定的半胱氨酸蛋白酶样蛋白，由基因编码，该基因在 III-E 型基因座中经常发现，这表明 III-E 型系统中的核糖核酸酶和蛋白酶活性之间存在功能联系。在这里，我们证明了靶 RNA 的识别会刺激 Csx29 的蛋白水解活性，而蛋白质 Csx30 是内源性底物。更有趣的是，虽然同源靶 RNA 的识别会激活 Csx29，但具有互补 3'反标签序列的非同源靶 RNA 会抑制酶活性。Csx30 可以与 sigma 因子 RpoE 结合，这可能会在蛋白水解切割后引发应激反应。结合生化和结构研究，我们阐明了 Csx29 靶 RNA 指导的蛋白水解活性的机制。我们的工作将指导进一步利用这个简单的 RNA 靶向系统进行 RNA 和蛋白质相关应用的开发。
