Potential role for snoRNAs in PKR activation during metabolic stress

Abstract

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.PKR | snoRNA | metabolic stress | phosphorylation | RNA-binding protein

Activation of PKR by short stem–loop RNAs containing single-stranded arms

“…snoRNAs, which contain short duplexes interspersed with singlestranded regions, were recently reported to activate PKR. In some cases activation is dependent on a 5 ′ -ppp, whereas others are active in 5 ′ -OH and 5 ′ -p states (Youssef et al 2015), suggesting that the structural context can modulate the triphosphate dependence.…”

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress

“…No reports exist in the literature to suggest that dsRNA is present under this stress condition, so we hypothesized an alternative method of PKR activation. Previous studies have elegantly shown the dependence of PKR activation by poly(I·C) in MEF cells genetically deficient in PKR and stably reconstituted with either wild-type human PKR (hPKR) or mutant human PKR (Mut hPKR) (56). We obtained cells in which residues lysine-64 in the dsRNA-binding motif (dsRBM) I and lysine-154 in the dsRNA-binding domain II were mutated to glutamines, impairing the binding of double-stranded RNA.…”

Identification of the long, edited dsRNAome of LPS-stimulated immune cells

“…In mammals, multiple dsRBPs activate the inflammatory response upon interaction with dsRNA, including toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), EIF2AK2 (also known as PKR), and DEXD/H-box helicase 58 (DDX58, also known as RIG-I) (de Faria et al 2013). These proteins were classically defined by their roles in initiation of an antiviral response, but recent data suggest that they are also activated by aberrantly regulated endogenous RNA (Hartner et al 2009;Pan et al 2011;Mannion et al 2014;Liddicoat et al 2015;Youssef et al 2015). Although the endogenous dsRNA pool is not well defined, host systems that regulate levels of dsRNA in the cell are known.…”

“…Several other host receptors also bind specific endogenous dsRNA ligands under sterile conditions, including TLR3 and EIF2AK2, again emphasizing the importance of dsRNA in aberrant inflammation (Cavassani et al 2008;Green et al 2012;Youssef et al 2015). In these cases, a few endogenous ligands are known, including U2 RNA, snoRNAs, and tRNAs (Bernard et al 2012;Youssef et al 2015), but the complete pool of long dsRNA remains elusive.…”