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. 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.