Initiation of a ZAKa-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L

RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L modulates host transcriptome through its endoribonuclease activity. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells. Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAK?. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38? are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes. Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAK? kinase cascade leading to proinflammatory signaling and apoptosis. Overall design: To examine the impact of 2-5A induced RNase L activity on transcriptome, the WT or RNaseLKO bone-marrow-derived macrophages will be mock transfected or transfected with 2-5A or its analog A3; 2 hours after transfection, the cells were harvested and the total mRNA were purified for total RNA sequencing; the mRNA from two sets of experiments were sequenced;the RNA-seq data were then analysed for the gene expression profiling; the gene expression profiling between Mock transfected and 2-5A/A3 transfected cells were compared as well as the difference between WT and RNaseLKO cells were analysed.