Bacterial Schlafens mediate anti-phage defense [3'-end RNA-seq]

Human Schlafen proteins restrict viral replication by cleaving tRNA and suppressing protein synthesis. The ribonuclease domain of Schlafen proteins is highly conserved and found in all three domains of life. However, its function in prokaryotes has not been determined experimentally. Here, we demonstrate that Schlafen nucleases are widespread in bacteria and mediate anti-phage defense. Bacterial Schlafen nucleases are frequently fused to a variety of domains that we predict to sense phage infection. We show that single-component phage defense, consisting of Schlafen nuclease fused to an immunoglobulin-like domain, recognizes T5-like phage tail assembly chaperones and cleaves bacterial and viral tRNA, triggering abortive infection. This work demonstrates that the Schlafen nuclease domain is an ancient, functionally and mechanistically conserved immune effector leveraged for antiviral defense across the tree of life. Overall design: to determine RNA cleaved by phage-activated bacterial Schlafen nuclease, we extracted total RNA from infected cells expressing active Schlafen nuclease or its catalytically inactive mutant. RNA was polyadenylated, sheared with ultrasound, reverse-transcribed with a polyT primer, and PCR amplified. Resulting amplicons were sequenced with Oxford Nanopore.

人类Schlafen蛋白可通过切割转运RNA（tRNA）并抑制蛋白质合成，从而限制病毒复制。Schlafen蛋白的核糖核酸酶结构域（ribonuclease domain）高度保守，广泛存在于三大生命域中。然而，其在原核生物中的功能尚未通过实验得以阐明。本研究证实，Schlafen核酸酶广泛分布于细菌中，并介导抗噬菌体防御功能。细菌来源的Schlafen核酸酶常与多种结构域融合，我们推测这些结构域可感知噬菌体感染。我们发现，由Schlafen核酸酶与免疫球蛋白样结构域融合构成的单组分噬菌体防御系统，可识别T5样噬菌体的尾部组装伴侣蛋白，并切割细菌与病毒tRNA，从而触发流产感染。本研究表明，Schlafen核酸酶结构域是一类古老且在功能与机制上均保守的免疫效应因子，被全生命界的生物用于抗病毒防御。
实验设计：为鉴定被噬菌体激活的细菌Schlafen核酸酶所切割的RNA，我们从表达有活性Schlafen核酸酶或其催化失活突变体的感染细胞中提取总RNA。将提取的RNA进行多聚腺苷酸化处理，随后通过超声进行片段化，以寡聚dT引物进行反转录，再通过聚合酶链式反应（PCR）扩增。所得扩增产物采用牛津纳米孔（Oxford Nanopore）技术进行测序。