title: 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 (PKRWT) or PKR with a point mutation in each dsRNA-binding motif (PKRRM). 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 PKRWT samples after PA treatment, but not in the PKRRM 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.
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112;
authors.forename: Osama A
authors.initials: OA
authors.lastname: Youssef
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112;
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Safran
authors.affiliation: Divisions of Endocrinology and Developmental Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Takahisa
authors.identifier: http://orcid.org/0000-0003-4922-4569
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112; and.
authors.forename: David A
authors.initials: DA
authors.lastname: Nix
authors.affiliation: Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard School of Public Health, Boston, MA 02115 bbass@biochem.utah.edu ghotamis@hsph.harvard.edu.
authors.forename: Gökhan S
authors.initials: GS
authors.lastname: Hotamisligil
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112; bbass@biochem.utah.edu ghotamis@hsph.harvard.edu.
authors.forename: Brenda L
authors.initials: BL
authors.lastname: Bass
mesh_terms: D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D047468:Immunoprecipitation; D051379:Mice; D019308:Palmitic Acid / Q000494:pharmacology; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D015203:Reproducibility of Results; D013312:Stress, Physiological / Q000187:drug effects; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D020537:RNA, Small Nucleolar; D019308:Palmitic Acid; D019892:eIF-2 Kinase
keywords: PKR; RNA-binding protein; metabolic stress; phosphorylation; snoRNA
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