Potential role for snoRNAs in PKR activation during metabolic stress

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR WT) or PKR with a point mutation in each dsRNA-binding motif (PKR RM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV–cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR WT samples after PA treatment, but not in the PKR RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress. Significance Animals respond to stress in many ways, including initiating cell death to eliminate damaged cells. Protein kinase RNA-activated (PKR) is a protein that senses stress, and it promotes cell death by phosphorylating eIF2α to block protein synthesis in damaged cells. PKR is activated by metabolic stress, such as that associated with obesity, and this activation depends on its RNA-binding domain. Here we investigated whether endogenous RNA triggers PKR activation in response to lipid exposure. Our results indicate that a noncoding RNA, the small nucleolar RNA (snoRNA), binds PKR during cellular metabolic stress, and multiple experiments suggest snoRNAs also activate PKR during metabolic stress. snoRNAs have established roles in RNA modification, and our studies suggest they have additional roles in metabolic stress.