Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response. Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response

During viral infection, several dsRNA sensors are activated in the cell and trigger changes in gene expression. One of these sensors activates a generic endonuclease, RNase L, that cleaves many types of RNA in the cell. However, how the resultant widespread RNA decay affects gene expression is not fully understood. Here we found that gene expression changes caused by activating dsRNA sensing pathways are tuned by RNase L activation, pointing to an intricate antiviral response where multiple inputs are integrated to create an optimized output. We show that RNA fragmentation induces the activation of the Ribotoxic Stress Response, potentially through ribosome collisions. The p38 and JNK pathways that are actuated as part of this response promote outcomes that likely inhibit the virus, such as programmed cell death. We also show that RNase L appears to limit the translation of genes that are regulated by another dsRNA-induced pathway, the Integrated Stress Response. Intriguingly, we found the activity of another generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage events can directly evoke an antiviral program. Overall design: To measure changes in the transcriptome under activation of RNase L, but not other components of the innate immune response, we performed RNA-seq on wild type (WT) and RNASEL KO cells that were transfected with 2-5A. Then, we analyzed differential expression (DE) patterns in 2-5A treated vs untreated cells by DEseq2. To investigate whether activation of RNase L modulates the effects of other pathways that are typically activated during infection by other dsRNA sensors, we simultaneously activated multiple pathways and tested RNase L’s role by transfecting WT and RNASEL KO A549 cells with poly I:C, a dsRNA mimic, followed by RNA-seq analysis. Then, we analyzed differential expression analysis of poly I:C treated vs untreated cells with DEseq2. To investigate how global RNA cleavage changes genes expression, we electroporated a generic RNase, RNase A, in RNASEL KO A549 cells and performed RNA-seq after 4.5 hours. Differential gene expression analysis between KO cells transfected wth either RNAse A or BSA (conotrols) was performed with DESeq2.

在病毒感染过程中，细胞内多种双链RNA（dsRNA）感受器会被激活，并触发基因表达的改变。其中一类感受器会激活一种通用型核酸内切酶——核糖核酸酶L（RNase L），该酶可切割细胞内多种类型的RNA。然而，由此产生的广泛RNA降解如何影响基因表达，目前尚未完全阐明。本研究发现，激活dsRNA感知通路所引发的基因表达变化，可通过RNase L的激活进行调控，这揭示了一种复杂的抗病毒应答机制：机体可整合多种输入信号，以生成优化的应答输出。研究表明，RNA片段化可诱导核糖体毒性应激反应（Ribotoxic Stress Response）的激活，这一过程可能通过核糖体碰撞实现。作为该应激反应的一部分被激活的p38与c-Jun氨基末端激酶（JNK）通路，可促进有助于抑制病毒的效应，例如细胞程序性死亡。此外，研究发现RNase L似乎可抑制由另一种dsRNA诱导通路——整合应激反应（Integrated Stress Response）所调控的基因的翻译过程。有趣的是，本研究发现另一种通用型核酸内切酶——核糖核酸酶A（RNase A）的活性，可重现激活态RNase L所介导的诸多分子表型，这证明了广泛的RNA切割事件可直接诱发抗病毒应答程序。实验设计：为了检测仅在RNase L激活、而非天然免疫其他组分激活的情况下的转录组变化，我们对转染了2-5A的野生型（WT）及RNASEL敲除（KO）细胞进行了RNA测序（RNA-seq）。随后，我们通过DESeq2分析了2-5A处理组与未处理组细胞间的差异表达（DE）模式。为了探究RNase L的激活是否会调控其他通常由其他dsRNA感受器在感染过程中激活的通路的效应，我们同时激活了多条通路，并通过将dsRNA模拟物聚肌胞苷酸（poly I:C）转染至野生型及RNASEL敲除A549细胞中，以验证RNase L的作用，随后进行了RNA-seq分析。随后，我们通过DESeq2对poly I:C处理组与未处理组细胞进行了差异表达分析。为了探究全局RNA切割如何改变基因表达，我们将通用型核糖核酸酶RNase A电转至RNASEL敲除A549细胞中，并在4.5小时后进行了RNA-seq分析。我们通过DESeq2对转染了RNase A或牛血清白蛋白（BSA，对照）的敲除细胞进行了差异基因表达分析。