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A double-stranded-RNA response program important for RNA interference efficiency

Department of Physiology, Room ND13.214A, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040, USA.
Molecular and Cellular Biology (Impact Factor: 5.04). 07/2007; 27(11):3995-4005. DOI: 10.1128/MCB.00186-07
Source: PubMed

ABSTRACT When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.

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    • "QDE-2 is an Argonaute protein and the core component of the RISC complex associated with siRNA [16] [17] [18]. Neurospora DCL-2 is responsible for most of the siRNA-generating activity [19]. "
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    ABSTRACT: In the filamentous fungus Neurospora, the production of dsRNA can elicit a dsRNA-induced transcriptional response similar to the interferon response in vertebrates. However, how fungi sense the expression of dsRNA and activate gene expression is unknown. In this study, we established a dsRNA response reporter system in Neurospora crassa. Using the dsRNA-activated RNA-dependent RNA polymerase gene rrp-3 promoter, we created an expression construct (pRRP-3::Myc-Al-1) and introduced it into al-1(KO) mutant. The test dsRNA efficiently induced pRRP-3::Myc-Al-1 expression in the al-1(KO) mutant, resulting in conidia color switching from white to yellow. These results confirm that the dsRNA response is regulated at the transcriptional level and this reporter system can be used for future studies in dsRNA response in filamentous fungi.
    FEBS letters 02/2011; 585(6):906-12. DOI:10.1016/j.febslet.2011.02.024 · 3.34 Impact Factor
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    • "Recent studies have suggested that RNA silencing may also play an important role in N . crassa DNA repair . It was shown that qde - 2 levels were induced by DNA damage ( Lee et al . , 2009 ) . Since earlier experiments suggested that dsRNA directly induces qde - 2 ( Choudhary et al . , 2007 ) , it was hypothesized that DNA damage somehow results in the production of dsRNA ( Lee et al . , 2009 ) . Consistent with this hypothesis , Lee et al . ( 2009 ) identified a new class of 21 nt sRNAs , termed qiRNAs , for their association with Qde - 2 under DNA damaging conditions , which mapped primarily to the rDNA locus . They also"
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    ABSTRACT: RNA silencing is an evolutionarily conserved mechanism in eukaryotic organisms induced by double-stranded RNA (dsRNA) and plays an essential role in regulating gene expression and maintaining genome stability. RNA silencing occurs at both posttranscriptional levels through sequence-specific RNA degradation or translational repression and at transcriptional levels through RNA-directed DNA methylation and/or heterochromatin formation. RNA silencing pathways have been relatively well characterized in plants and animals, and are now also being widely investigated in diverse fungi, some of which are important plant pathogens. This review focuses primarily on the current understanding of the dsRNA-mediated posttranscriptional gene silencing processes in fungi, but also discusses briefly the known gene silencing pathways that appear to be independent of the RNA silencing machineries. We review RNA silencing studies for a variety of fungi and highlight some of the mechanistic differences observed in different fungal organisms. As RNA silencing is being exploited as a technology in gene function studies in fungi as well as in engineering anti-fungal resistance in plants and animals, we also discuss the recent progress towards understanding dsRNA uptake in fungi. KeywordsRNA silencing-gene silencing-heterochromatin formation-fungi-small interfering RNA-micro-RNA
    12/2010; 5(6):478-494. DOI:10.1007/s11515-010-0550-3
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    ABSTRACT: Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms. Solun normaali toiminta perustuu erilaisten proteiinimolekyylien oikeaan määrään, rakenteeseen sekä aktiivisuuteen. Geenit sisältävät ohjeet proteiinien synteesiin, mutta geenien ilmentymistä säätelee valtavan monimutkainen ja yhä laajalti tuntematon biokemiallinen verkosto. Vasta hiljattain on ymmärretty, että erilaiset RNA-molekyylit ohjaavat geenien ilmentymistä useimmissa eliöissä. Etenkin kaksijuosteisen RNA:n on havaittu säätelevän toisten RNA-molekyylien määrää ja aktiivisuutta hyvinkin täsmällisesti. Ilmiötä kutsutaan RNA-tason geneettiseksi hiljentämiseksi, ja se on erittäin hyvin säilynyt aitotumallisten eliöiden evoluutiossa. -- Tässä työssä tutkitaan Neurospora crassa sienestä eristettyä QDE-1 proteiinia, joka on RNA:ta templaattinaan käyttävä RNA-polymeraasi (RdRP). QDE-1 on tärkeässä asemassa siirtogeenisten sekä hyppivien elementtien vaimentamisessa. On ajateltu, että QDE-1 tunnistaa siirtogeenisistä alueista peräisin olevat RNA-molekyylit ja muuttaa ne kaksijuosteiseksi RNA:ksi. Tämä kaksijuosteinen RNA prosessoidaan pienemmiksi siRNA-molekyyleiksi, jotka puolestaan ohjaavat siirtogeenisten RNA-molekyylien hajotusta. Väitöskirjassa osoitetaan, että QDE-1:lla on aikaisemmin tunnettujen ominaisuuksien lisäksi monia muita katalyyttisia aktiivisuuksia. Näistä tärkein on DNA:sta riippuvainen RNA-polymeraasiaktiivisuus (DdRP), joka osoittautui aiemmin tunnettua RdRP-aktiivisuutta paljon merkittävämmäksi. Lisäksi havaittiin, että QDE-1 aktivoituu solun DNA:n vaurioituessa ja että tämä aktivoituminen johtaa uudentyyppisten pienten RNA-molekyylien (qiRNA) synteesiin. Edelleen osoitettiin, että QDE-1 muodostaa kompleksin kahden muun proteiinin (QDE-3, RPA) kanssa, mikä muokkaa QDE-1:n toimintaa. Väitöskirjassa esitetyt tulokset uudistavat käsitystämme Neurospora crassa sienen geenien ilmentymisen säätelystä. Uuden, väitöskirjassa esitetyn mallin mukaan QDE-1 on aktiivinen soluliman lisäksi myös tumassa. Se voi käyttää templaattinaan sekä DNA:ta että RNA:ta, ja DNA:n vaurioituminen johtaa uudentyyppiseen biokemialliseen vasteeseen. Näillä tuloksilla on merkitystä myös laajemmin, sillä samankaltaisia RNA-polymeraaseja on löydetty miltei kaikista tutkituista aitotumallisista eliöistä. Vastaavat säätelymekanismit saattavat siis vallita myös kasveissa ja eläimissä.
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