Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines

Billy, Éric; Brondani, Vincent; Zhang, Haidi; Müller, Ulrich; Filipowicz, Witold

doi:10.1073/pnas.261562698

Cited by 362 publications

(235 citation statements)

References 41 publications

(77 reference statements)

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“…Similarly, dsRNAi was not affected by inducible expression of FLAG‐RIG‐I or FLAG‐MDA5 (Fig EV5B–D), confirming the observations in vitro (Fig 4C and D) that full‐length LGP2 but not the other members of the RLR family have the ability to block Dicer. Of note, doxycycline‐inducible expression of LGP2 did not block dsRNAi in an embryonic carcinoma cell line (F9) that was previously shown to efficiently process dsRNA into siRNAs whilst being refractory to activation of the IFN response (Fig EV6A and B; Billy et al , 2001). This observation indicates that LGP2 may work in concert with other ISGs or IFN‐controlled mechanisms absent in F9 cells to suppress dsRNAi.…”

Section: Resultsmentioning

confidence: 81%

“…In contrast, others have found low abundance of viRNAs in mammalian somatic cells infected with various viruses, and only a modest effect of Dicer deficiency on viral replication, suggesting that RNAi is not an active mechanism of antiviral defence in most mammalian cell types (Parameswaran et al , 2010; Girardi et al , 2013; Backes et al , 2014; Bogerd et al , 2014; Schuster et al , 2017). RNAi may be particularly important in undifferentiated mammalian cells, and clear evidence of the existence of endogenous RNAi, dsRNA‐mediated RNAi (dsRNAi), or antiviral RNAi has been documented in oocytes, embryonic teratocarcinoma cell lines and mouse embryonic stem cells (mESCs), respectively (Billy et al , 2001; Flemr et al , 2013; Maillard et al , 2013). In mESCs, viral infection leads to the Dicer‐dependent emergence of viRNAs that associate with Ago2 and inhibit replication of a VSR‐deficient homologous virus (Maillard et al , 2013).…”

Section: Introductionmentioning

confidence: 99%

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The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

et al. 2018

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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 antiviral 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 show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. 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. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.

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Section: Resultsmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

et al. 2018

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“…As mentioned previously, failure to document this process in the context of viral infection is complicated by the possibility that the virus encodes VSRs (Haasnoot et al , 2007; Wu et al , 2010). Therefore, we designed a virus‐free assay to measure long dsRNA‐mediated gene silencing at the single‐cell level and tested it initially in mouse embryonic stem cells (mESCs), in which the presence of dsRNAi and antiviral RNAi has been reported (Billy et al , 2001; Yang et al , 2001; Paddison et al , 2002; Maillard et al , 2013). mESCs stably expressing a GFP reporter gene were transfected with in vitro synthesised Cy5‐labelled long dsRNA corresponding to the first 200 nt of either the GFP (dsRNA‐GFP) or, as a control, the Renilla luciferase (dsRNA‐ RL ) coding sequences; GFP expression in live single Cy5‐positive (transfected) cells was then measured by flow cytometry (Fig 1A and B).…”

Section: Resultsmentioning

confidence: 99%

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

et al. 2016

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RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.

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“…Dicer is another RNaseIII-type enzyme, and it cleaves pre-miRNAs in the cytoplasm (Billy et al, 2001;Grishok et al, 2001;Hutva´gner et al, 2001;Ketting et al, 2001;Provost et al, 2002;Zhang et al, 2002). Most Dicer proteins possesses an approximately 130-amino-acid-long PAZ domain (Figure 3), which preferentially binds to single-stranded 3 0 ends of doublestranded RNAs (Lingel et al, 2003;Song et al, 2003;Yan et al, 2003;Ma et al, 2004).…”

Section: Dicer Cleavage Of Pre-mirnasmentioning

confidence: 99%

Principles of micro-RNA production and maturation

2006

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Micro-RNAs (miRNAs) are a class of approximately 22-nucleotide non-coding RNAs expressed in multicellular organisms. They are first transcribed in a similar manner to pre-mRNAs. The transcripts then go through a series of processing steps, including endonucleolytic cleavage, nuclear export and a strand selection procedure, to yield the single-stranded mature miRNA products. The transcription and processing of miRNAs determines the abundance and the sequence of mature miRNAs and has important implications for the function of miRNAs.

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Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines

Cited by 362 publications

References 41 publications

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

Principles of micro-RNA production and maturation

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