An isoform of Dicer protects mammalian stem cells against multiple RNA viruses
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE173946 | GEO | 2021/07/09
REPOSITORIES: GEO
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Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals. Infection of wild-type or mutant mouse ES cells and analysis of small RNAs from total extracts or immunoprecipitated components of the RNAi pathway
2013-10-10 | E-GEOD-43153 | biostudies-arrayexpress
Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals.
2013-10-10 | GSE43153 | GEO
Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.
2018-01-17 | PXD008364 | Pride
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells
2013-01-31 | E-GEOD-28957 | biostudies-arrayexpress
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics.
2013-01-31 | GSE28957 | GEO
Project description:RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates, however whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner, loaded into AGO, and were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer-deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.
2017-08-01 | GSE99252 | GEO
Project description:In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1-expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
2024-01-26 | GSE241798 | GEO
Project description:In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1-expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
2024-01-26 | GSE241796 | GEO
Project description:In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1-expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
2024-01-26 | GSE241797 | GEO
Project description:The function and mechanism of the interferon-regulated antiviral responses have been extensively characterized. Recent studies have demonstrated induction of antiviral RNA interference (RNAi) in somatic cells against several mammalian RNA viruses rendered incapable of RNAi suppression. However, little is known about Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNAs) in these cells active in the type I interferon response. Here we show that the dsRNA precursors of influenza vsiRNAs were processed more efficiently than cellular precursor microRNA hairpins by wild-type human Dicer expressed de novo in Dicer-knockout somatic cells. We found that infection of two strains of suckling mice with wild-type Nodamura virus (NoV) was associated with production of silencing-active vsiRNAs and direct sequestration of duplex vsiRNAs by its RNAi suppressor protein B2. Our findings from in vivo infection with Sindbis virus recombinants expressing NoV B2 or carrying a vsiRNA-targeted insert provide evidence for an antiviral function of the induced RNAi response. Interestingly, NoV infection induces expression of RIG-I-like receptor LGP2 to inhibit vsiRNA biogenesis and promote virulent infection in suckling mice. Our findings together reveal efficient Dicer processing of vsiRNA precursors in interferon-competent somatic cells and suckling mice in contrast to synthetic long dsRNA examined previously by in vitro dicing.
2021-07-28 | GSE155146 | GEO