Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response [ribosome profiling]

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 test if the activity of any generic endonuclease leads to increased translation initiation at alternative ORFs within the 3' UTR of genes, we electroporated RNase A into RNASEL KO A549 cells and performed ribosome profiling on those same samples after 4.5 hours. In addition, to assess changes in translation due to activation of the dsRNA sensor pathway involving RNase L, RNASEL WT and KO A549 cells were transfected with either 2-5A or poly(I:C) followed by ribosomal profiling after 4.5 hours.