Abstract:Double-stranded RNA (dsRNA) is a danger signal that triggers endonucleolytic degradation of RNA inside infected and stressed mammalian cells. This mechanism inhibits growth and ultimately removes problematic cells via apoptosis. To elucidate the molecular functions of this program and understand the connection between RNA cleavage and programmed cell death, we visualized dsRNA-induced degradation of human small RNAs using RtcB ligase-assisted RNA sequencing (RtcB RNA-seq). RtcB RNA-seq revealed strong cleavage… Show more
“…In addition to cleaving rRNA, RNase L was recently reported to strongly and site-specifically cleave tRNA-His at position 36 located in the anti-codon loop. The site-specific tRNA-His cleavage was readily observed using a quantitative RT-PCR assay in IFN-treated MAVS-ADAR1 DKO, but not in IFN-treated cells, which lack RNase L activity in the above assays: MAVS KO and RNASEL-ADAR1 DKO cells (Figure 7c) (Donovan et al, 2016). 10.7554/eLife.25687.020Figure 6.Interferon (IFN) and interferon stimulated gene (ISG) expression following IFN treatment of ADAR1 KO cells.Cells were treated or mock treated with 10 U/ml of IFN-α and 24 hr post treatment, lysed and RNA extracted.…”
Section: Resultsmentioning
confidence: 99%
“…Based on these results, we propose that RNase L has a predominant role in promoting cell death by dsRNA ( Figure 1 ). It has been shown recently that RNase L site-specifically cleaves tRNAs and Y-RNAs, and that depletion of the Y-RNA RNY1 contributes to apoptosis ( Donovan et al, 2016 ).…”
ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway for endogenous dsRNA that leads to cell death.DOI:
http://dx.doi.org/10.7554/eLife.25687.001
“…In addition to cleaving rRNA, RNase L was recently reported to strongly and site-specifically cleave tRNA-His at position 36 located in the anti-codon loop. The site-specific tRNA-His cleavage was readily observed using a quantitative RT-PCR assay in IFN-treated MAVS-ADAR1 DKO, but not in IFN-treated cells, which lack RNase L activity in the above assays: MAVS KO and RNASEL-ADAR1 DKO cells (Figure 7c) (Donovan et al, 2016). 10.7554/eLife.25687.020Figure 6.Interferon (IFN) and interferon stimulated gene (ISG) expression following IFN treatment of ADAR1 KO cells.Cells were treated or mock treated with 10 U/ml of IFN-α and 24 hr post treatment, lysed and RNA extracted.…”
Section: Resultsmentioning
confidence: 99%
“…Based on these results, we propose that RNase L has a predominant role in promoting cell death by dsRNA ( Figure 1 ). It has been shown recently that RNase L site-specifically cleaves tRNAs and Y-RNAs, and that depletion of the Y-RNA RNY1 contributes to apoptosis ( Donovan et al, 2016 ).…”
ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway for endogenous dsRNA that leads to cell death.DOI:
http://dx.doi.org/10.7554/eLife.25687.001
“…2-5A binds to the ankyrinrepeat (ANK) domain of RNase L and promotes its oligomerization and formation of a dimeric endoribonuclease active site [21][22][23] . This dimer further assembles into high-order oligomers 24 that cleave viral RNAs 16,18 and all components of the translation apparatus, including mRNAs 25 , tRNAs 26 and 28S/18S rRNAs 27,28 . The resulting action of RNase L inhibits global translation, which puts all proteins, including IFNs, at risk of arrest during a cellular response to dsRNA.…”
Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here we developed a biosensor for 2',5'oligoadenylate (2-5A), the natural activator of RNase L. We found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However,
translation-arrested cells still transcribed IFN-stimulated genes (ISGs) and secretedIFNs of types I and III (IFN-β and IFN-). Our data suggests that IFNs escape from the action of RNase L on translation. We propose that 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.
SignificanceRNase L is a mammalian enzyme that can stop global protein synthesis during interferon response. Cells must balance the need to make interferons (which are proteins) with the risk to lose cell-wide translation due to RNase L. This balance can most simply be achieved if RNase L was activated late in the interferon response. However, we show by engineering a biosensor for the RNase L pathway, that on the contrary, RNase L activation precedes interferon synthesis.Further, translation of interferons evades the action of RNase L. Our data suggest that RNase L facilitates a switch of protein synthesis from homeostasis to specific needs of innate immune signaling.. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/476341 doi: bioRxiv preprintThe 2-5A system is also a surveillance pathway for endogenous dsRNAs from mammalian genomes. Cells with adenosine deaminase 1 (ADAR1) deficiency accumulate self-dsRNA that promotes 2-5A-driven apoptosis 8 . 2-5A synthesis in the presence of low amounts of endogenous dsRNAs does not cause cell death, but functions as a suppressor of adhesion, proliferation, migration, and prostate cancer metastasis 17,18 . Additionally, activation of the 2-5A system also blocks secretion of milk . CC-BY 4.0 International license is made available under a
“…2-5A binds to the ankyrin-repeat (ANK) domain of RNase L and promotes its oligomerization and the formation of a dimeric endoribonuclease active site (21)(22)(23). This dimer further assembles into high-order oligomers (24) that cleave viral RNAs (16,18) and all components of the translation apparatus, including mRNAs (25), tRNAs (26), and 28S/18S rRNAs (27,28). The resulting action of RNase L inhibits global translation, which puts all proteins, including IFNs, at risk for arrest during a cellular response to dsRNA.…”
Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2′,5′-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system. interferon | translation reprogramming | 2-5A
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