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

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examinedHLAassociations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominantHLA-DRB1*08:04andHLA-DRB1*11:02alleles were associated with overall SSc risk, and theHLA-DRB1*08:04allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, theHLA-DPB1*13:01andHLA-DRB1*07:01alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance ofHLAin defining autoantibody subtypes. The association of theHLA-DPB1*13:01allele with the ATA+subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence andHLA-DPB1*13:01allele frequency in multiple populations was observed (r= 0.98,P= 3 × 10−6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/β allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link betweenHLAalleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

In situ colonization experiments were performed to study the pioneer populations of bacteria at Loihi Seamount, Hawaii. Over a ten-year sampling period, 41 microbial growth chambers (MGCs) were deployed and recovered in Pele's Pit and the surrounding area after short-term (4-10 days) and long-term (∼1-6 years) incubations in the flow of hydrothermal effluent. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the small subunit rRNA gene (SSU rDNA) revealed that the short-term MGC communities exhibited a low number of represented populations when compared to the long-term MGC communities and naturally occurring microbial mats. Cluster analysis of T-RFLP fingerprints showed the short-term MGC communities all had similar richness but were separated into three distinct groups with different arrays of colonizing populations. Clone library analysis showed that cooler vents (T ave = 40 • C) were primarily colonized by Mariprofundus ferrooxydans, a neutrophilic Fe-oxidizing ζ -Proteobacteria while warmer vents (T ave = 71 • C) were colonized by Sulfurimonas spp. and other sulfur-cycling members of the ε-Proteobacteria. Vents with an intermediate temperature (T ave = 51 • C) were colonized by representatives of both ζ -Proteobacteria and ε-Proteobacteria. Long-term MGC communities did not cluster with any of the short-term communities and exhibited higher richness, indicating a greater number of bacterial populations were able to colonize and grow in the long-term growth chambers.

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5 ′ ′ ′ ′ ′ terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5 ′ ′ ′ ′ ′ -phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5 ′ ′ ′ ′ ′ -triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5 ′ ′ ′ ′ ′ triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.