PACT-mediated regulation of endogenous dsRNA sensing by PKR
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1123226 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29380800_1.fastq.gz | Fastqsanger.gz | |||
| SRR29380800_2.fastq.gz | Fastqsanger.gz | |||
| SRR29380801_1.fastq.gz | Fastqsanger.gz | |||
| SRR29380801_2.fastq.gz | Fastqsanger.gz | |||
| SRR29380802_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein kinase R (PKR), an innate immune sensor for double-stranded RNA (dsRNA), is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a crucial and controversial role in the PKR pathway. We demonstrate that PACT is an essential negative regulator of PKR against endogenous dsRNA ligands like inverted repeat Alu RNAs, which satisfy PKR’s selectivity for long dsRNA and robustly activate PKR in the absence of PACT. PACT employs an unusual inhibitory mechanism sensitive to dsRNA length and concentration, inhibiting PKR through low-affinity interactions most effective when both are bound to the same dsRNA molecule. Consequently, PACT’s inhibition of PKR is more robust when dsRNA is longer and less abundant, with minimal effect on abundant or short dsRNA. Thus, PACT functions as a rheostat, setting the PKR activation threshold for long endogenous dsRNA without altering its inherent activity.
2024-06-16 | GSE269684 | GEO
Project description:Triple-negative breast cancer (TNBC), the deadliest breast cancer subtype, lacks broadly applicable targeted therapies. Induction of ‘viral mimicry’ through activation of viral double-stranded RNA (dsRNA) sensors has potential therapeutic applications for TNBC and other cancers. Suppressors of dsRNA sensing prevent sensing of endogenous dsRNAs and resulting autoimmunity. Depletion of the suppressor of dsRNA sensing ADAR1 causes activation of dsRNA sensors and cell death in many cancer cell lines. These ADAR1-dependent cells are generally also dependent on the dsRNA-binding protein PACT, which is highly expressed and essential in many TNBC cell lines. While PACT is known as an activator of the dsRNA sensor PKR, overexpression of PACT had no effect on activation of PKR in multiple breast cancer cell lines. Conversely, depletion of PACT in PACT-dependent cell lines caused robust activation of the dsRNA sensor PKR and cell death, in addition to induction of integrated stress response genes and NF-κB targets. These phenotypes were entirely dependent on PKR. Rescue experiments revealed that PACT dimerization and dsRNA binding is required to suppress PKR activation. While depletion of PACT alone in ADAR1/ PACT-independent cell lines had no effect on PKR activation, combined depletion of both PACT and ADAR1 in those cell lines caused robust PKR activation and cell death, supporting a partially redundant role for ADAR1 and PACT in suppression of dsRNA sensing. Taken together, these findings support a vital role for PACT in suppression of PKR activation, and highlight the therapeutic potential of targeting PACT to treat TNBC.
2025-06-03 | GSE298233 | GEO
Project description:Cancer cells evolve various mechanisms to overcome cellular stresses and maintain progression. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders in monitoring diverse stress signals and lead to cell proliferation inhibition and cell apoptosis in consequence. However, the regulation of PACT-PKR pathway in cancer cells is largely unknown. Herein, we identify that the long non-coding RNA (lncRNA) aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes cancer cell proliferation. Using large-scale CRISPRi functional screening of 971 cancer-associated lncRNAs, we find that DARS-AS1 is associated with significantly enhanced cancer cell proliferation. Accordingly, knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro and significantly reduces tumor growth in vivo. Mechanistically, DARS-AS1 directly binds to the activator domain of PACT and prevents PACT-PKR interaction, thereby decreasing PKR activation, eIF2α phosphorylation and inhibiting apoptotic cell death. Clinically, DARS-AS1 is broadly expressed across multiple cancers and the increased expression of this lncRNA indicates poor prognosis. This study elucidates the lncRNA DARS-AS1 directed cancer-specific modulation of the PACT-PKR pathway and provides a novel target for cancer prognosis and therapeutic treatment.
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Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments. HEK293 cells (human origin) were transiently transfected with hairpin dsRNA-expressing (MosIR) and control (pCag) plasmids.
2013-12-27 | E-GEOD-46959 | biostudies-arrayexpress
Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments.
2013-12-27 | GSE46959 | GEO
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.
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Project description:Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy.
2015-01-29 | GSE28993 | GEO
Project description:Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.
2015-01-29 | E-GEOD-28993 | biostudies-arrayexpress
Project description:PACT suppresses PKR activation through dsRNA binding and dimerization, and is a therapeutic target for triple-negative breast cancer
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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.
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