PKR senses nuclear and mitochondrial signals by interacting with endogenous double-stranded RNAs
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PROVIDER: PRJNA429340 | ENA |
REPOSITORIES: ENA
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Project description:Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation-sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.
2018-07-18 | GSE108986 | GEO
Project description:Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on Thr446 and Thr451 residues. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 aminoacids upstream the first double-stranded RNA binding domain (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding, suggesting a model where negative charges occuring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive, closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes
2021-05-10 | PXD022564 | Pride
Project description:The antiviral defense in vertebrates requires the innate immune system to sense foreign “non-self” nucleic acids while avoiding “self” nucleic acids, which is accomplished by an intricate system. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern from being recognized as viral dsRNA. Lack of RNA editing by ADAR1 enables activation of MDA5, a cytosolic dsRNA sensor, by cellular dsRNA. Additional RNA editing- independent functions of ADAR1 have been proposed, but the specific mechanism remains elusive. Here we demonstrate that RNA binding by ADAR1, independent of its editing activity, restricts the activation of PKR, another cytosolic dsRNA sensor, by cellular dsRNA. Mechanistically, the loss of ADAR1 editing caused MDA5 activation to induce interferon signaling, while a lack of ADAR1 protein or its dsRNA binding ability led to PKR activation, with subsequent stress granule formation and proliferation arrest. Based on these findings we rescued the Adar1−/− mice from embryonic lethality to adulthood by deleting both MDA5 and PKR, in contrast to the limited rescue of Adar1−/− mice by removing MDA5 or PKR alone. Our findings reveal a multifaceted contribution of ADAR1 in regulating the immunogenicity of “self” dsRNAs. Furthermore, ADAR1 is an immuno-oncology target for drug development, and the separation of ADAR1’s RNA editing and binding functions provides mechanistic insights for such developments.
2023-10-03 | GSE198386 | GEO
Project description:PKR is an interferon induced serine/threonine protein kinase, that is activated by double stranded RNA. PKR plays an important role in the antiviral defense by interferon. In addition to its role in translation, PKR participates in several signaling pathways to transcription. The goal of this experiment is to study the role of PKR in regulating gene expression in our NIH 3T3 inducible cell line, which could overexpress PKR wt protein after the removal of tetracycline (Donze O, Dostie J, Sonenberg N. (1999) Virology 256: 322-9).
2004-01-30 | E-MEXP-50 | biostudies-arrayexpress
Project description:Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene (ISG) expression. XRN1 deletion causes PKR pathway activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-b stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR, and how this interaction creates a vulnerability in cancer cells with an activated interferon cell state.
2024-04-17 | GSE248036 | GEO
Project description:Exon(s) back-splicing-generated circular RNAs as a group can suppress the double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing circular RNAs as potent PKR inhibitors. Here, we report that circular RNAs synthesized by permuted td introns from T4 bacteriophage and by Anabeana pre-tRNA group I intron induce an immune response. Mechanistically, autocatalytic splicing introduces extra ~74 nt and ~186 nt fragments (E1+E2+spacer) that form additional stable loop structures that likely provoke innate immune responses in circular RNA products. In contrast, circular RNAs produced by T4 RNA ligase without unwanted fragment exhibit little immunogenicity. Importantly, directly-ligated circular RNAs form short dsRNA-regions exhibit 103~106 fold higher efficiency than the reported chemical compounds C16 and 2-AP in suppressing PKR activation, highlighting the use of circular RNAs as novel inhibitors for PKR over-reaction diseases.
2021-12-23 | GSE183941 | GEO
Project description:LincRNA-EPS negatively regulates host antiviral immunity by antagonizing viral RNA interacting with PKR
| PRJNA795796 | ENA
Project description:Global analysis of gene expression in NIH3T3 cells over-expressing RNA-dependent serine/threonine protein kinase (PKR). Keywords: other
2004-09-22 | GSE682 | GEO
Project description:ADAR1 utilizes independent mechanisms to suppress MDA5 and PKR activation by cellular double-stranded RNA
| PRJNA814746 | ENA
Project description:PKR (Protein Kinase RNA-activated) 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 snoRNAs (small nucleolar RNAs) associate with PKR during metabolic stress. Our high-throughput sequence shows that snoRNAs are enriched in PKR_WT samples after palmitic acid treatment compared to mock-treated samples. MEF cells reconstituted with wild type PKR (PKR_WT) or PKR with a mutation in each dsRBM (PKR_RM) were mock- or palmitate-treated. RNAs associated with PKR were immunoprecipitated. IPed RNAs were extracted, cloned and sequenced. We performed differential expression to obtain RNAs enriched in PKRWT after palmitate treatment. We sequenced three different datasets of samples (A, B and C). Each A and B dataset contains three biological replicates. Dataset C contains a single sample.
2015-05-08 | E-GEOD-66540 | biostudies-arrayexpress