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Maria Eugenia Sarasquete - One of the best experts on this subject based on the ideXlab platform.
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impaired expression of Dicer drosha sbds and some micrornas in mesenchymal stromal cells from myelodysplastic syndrome patients
Haematologica, 2012Co-Authors: Carlos A Santamaria, Sandra Muntion, Beatriz Roson, Belen Blanco, Olga Lopezvillar, Soraya Carrancio, Fermin Sanchezguijo, Maria Diezcampelo, Stela Alvarezfernandez, Maria Eugenia SarasqueteAbstract:Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze Dicer1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS , a Dicer1- related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. Design and Methods Mesenchymal stromal cells from 33 bone marrow samples were evaluated. Dicer , DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. Results Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower Dicer1 (0.65±0.08 vs. 1.91±0.57; P =0.011) and DROSHA (0.62±0.06 vs . 1.38±0.29; P =0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs . 1.15±0.28; P =0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where Dicer1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.
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impaired expression of Dicer drosha sbds and some micrornas in mesenchymal stromal cells from myelodysplastic syndrome patients
Haematologica, 2012Co-Authors: Carlos Santamaria, Sandra Muntion, Beatriz Roson, Belen Blanco, Olga Lopezvillar, Soraya Carrancio, Fermin Sanchezguijo, Maria Diezcampelo, Stela Alvarezfernandez, Maria Eugenia SarasqueteAbstract:Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze Dicer1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS , a Dicer1- related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. Design and Methods Mesenchymal stromal cells from 33 bone marrow samples were evaluated. Dicer , DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. Results Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower Dicer1 (0.65±0.08 vs. 1.91±0.57; P =0.011) and DROSHA (0.62±0.06 vs . 1.38±0.29; P =0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs . 1.15±0.28; P =0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where Dicer1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.
Shinyi Lee Marzano - One of the best experts on this subject based on the ideXlab platform.
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mycoviruses as triggers and targets of rna silencing in white mold fungus sclerotinia sclerotiorum
Viruses, 2018Co-Authors: Pauline Mochama, Prajakta Jadhav, Achal Neupane, Shinyi Lee MarzanoAbstract:This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, Dicer genes (dcl-1, dcl-2, and both dcl-1/dcl-2) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double Dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2. Complementation of a single Dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both Dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.
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Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum
MDPI AG, 2018Co-Authors: Pauline Mochama, Prajakta Jadhav, Achal Neupane, Shinyi Lee MarzanoAbstract:This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, Dicer genes (dcl-1, dcl-2, and both dcl-1/dcl-2) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double Dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2. Complementation of a single Dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both Dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus
Petr Svoboda - One of the best experts on this subject based on the ideXlab platform.
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Production of small RNAs by mammalian Dicer
Pflügers Archiv: European Journal of Physiology, 2016Co-Authors: Eliska Svobodova, Jana Kubikova, Petr SvobodaAbstract:MicroRNA (miRNA) and RNA interference (RNAi) pathways employ RNase III Dicer for the biogenesis of small RNAs guiding post-transcriptional repression. Requirements for Dicer activity differ in the two pathways. The biogenesis of miRNAs requires a single Dicer cleavage of a short hairpin precursor to produce a small RNA with a precisely defined sequence, while small RNAs in RNAi come from a processive cleavage of a long double-stranded RNA (dsRNA) into a pool of small RNAs with different sequences. While Dicer is generally conserved among eukaryotes, its substrate recognition, cleavage, and biological roles differ. In Metazoa, a single Dicer can function as a universal factor for RNAi and miRNA pathways or as a factor adapted specifically for one of the pathways. In this review, we focus on the structure, function, and evolution of mammalian Dicer. We discuss key structural features of Dicer and other factors defining Dicer substrate repertoire and biological functions in mammals in comparison with invertebrate models. The key for adaptation of Dicer for miRNA or RNAi pathways is the N-terminal helicase, a dynamically evolving Dicer domain. Its functionality differs between mammals and invertebrates: the mammalian Dicer is well adapted to produce miRNAs while its ability to support RNAi is limited.
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a retrotransposon driven Dicer isoform directs endogenous small interfering rna production in mouse oocytes
Cell, 2013Co-Authors: Matyas Flemr, Radek Malik, Vedran Franke, Jana Nejepinska, Radislav Sedlacek, Kristian Vlahovicek, Petr SvobodaAbstract:Summary In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (Dicer O ). Dicer O lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (Dicer S ). Unlike Dicer S , Dicer O efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the Dicer O isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of Dicer O and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline.
Pauline Mochama - One of the best experts on this subject based on the ideXlab platform.
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mycoviruses as triggers and targets of rna silencing in white mold fungus sclerotinia sclerotiorum
Viruses, 2018Co-Authors: Pauline Mochama, Prajakta Jadhav, Achal Neupane, Shinyi Lee MarzanoAbstract:This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, Dicer genes (dcl-1, dcl-2, and both dcl-1/dcl-2) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double Dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2. Complementation of a single Dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both Dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.
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Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum
2018Co-Authors: Pauline Mochama, Prajakta Jadhav, Achal Neupane, Shin-yi Lee MarzanoAbstract:This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by probing wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a circular DNA virus. Key silencing-related genes, specifically Dicers, were disrupted in order to dissect the RNA silencing pathway and provide useful information on fungal control. Dicers Dcl-1, Dcl-2, and both Dcl-1/Dcl-2- genes were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, susceptibility to viral infection, and small RNA accumulation compared to the wild-type strain. Disruption of Dcl-1 or Dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double Dicer mutant strain exhibited slower growth. To examine the effect of viral infection on strains containing null-mutations of Dcl-1, Dcl-2 or both genes, mutants were transfected with full-length RNA transcripts of a hypovirus SsHV2L and copies of a single-stranded DNA mycovirus- SsHADV-1 as a synthetic virus. Results indicate that the ΔDcl-1/Dcl-2 double mutant which was slow growing without virus infection exhibited much more severe debilitation following virus infection. Altered colony morphology including: reduced pigmentation, significantly slower growth, and delayed sclerotial formation. Additionally, there is an absence of virus-derived small RNAs in the virus-infected ∆Dcl-1/Dcl-2 mutant compared to the virus-infected wild-type strain which displays a high percentage of virus-derived small RNA. The findings of these studies suggest that if both Dicers are silenced, invasive nucleic acids which include mycoviruses ubiquitous in nature- can greatly debilitate the virulence of fungal plant pathogens.
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Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum
MDPI AG, 2018Co-Authors: Pauline Mochama, Prajakta Jadhav, Achal Neupane, Shinyi Lee MarzanoAbstract:This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, Dicer genes (dcl-1, dcl-2, and both dcl-1/dcl-2) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double Dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2. Complementation of a single Dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both Dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus
Jennifer A. Doudna - One of the best experts on this subject based on the ideXlab platform.
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a conserved mechanism of rna substrate recognition and cleavage by fungal Dicers
Biophysical Journal, 2014Co-Authors: Mary Anne Kidwell, Jessica M Chan, Jennifer A. DoudnaAbstract:Dicer is a central enzyme for processing small RNAs in RNA interference. It is able to cleave both hairpin and long double-stranded RNA precursors into microRNAs and small interfering RNAs respectively. While humans have one Dicer that is able to process both substrates, many organisms have multiple copies. Understanding how organisms with two Dicers chose specific RNAs for cleavage could give insight into how the human protein functions.We have turned to the thermophilic fungus Sporotrichum thermophile as a model system to purify stable Dicers for biochemical dissection. While the function of these two Dicers was thought to be partially redundant, our results show that they cleave different RNA substrates, similar to what is observed in the Drosophila melanogaster system. S. thermophile Dicer-1 dices hairpin precursor microRNAs faster than long double-stranded RNAs while S. thermophile Dicer-2 can only cleave perfect RNA duplexes accurately with the requirement for ATP. Removal of the helicase domain from Dicer-2 relieves the requirement of ATP for cleavage and allows the enzyme to process hairpin substrates. In addition, only Dicer-2 shows robust ATP hydrolysis in the presence of dsRNA and partial stimulation in the presence of single-stranded RNA or an RNA/DNA hybrid. The conserved function of these two Dicers may indicate a common mechanism used by eukaryotes to process small RNAs.
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Structure of Dicer and Mechanistic Implications for RNAi
Cold Spring Harbor symposia on quantitative biology, 2006Co-Authors: Ian J. Macrae, Kaihong Zhou, W.z. Cande, Jennifer A. DoudnaAbstract:Dicer is a specialized ribonuclease that processes double-stranded RNA (dsRNA) into small RNA fragments about 25 nucleotides in length during the initiation phase of RNA interference (RNAi). We previously determined the crystal structure of a Dicer enzyme from the diplomonad Giardia intestinalis and proposed a structural model for dsRNA processing. Here, we provide evidence that Dicer is composed of three structurally rigid regions connected by flexible hinges and propose that conformational flexibility facilitates dsRNA binding and processing. We also examine the role of the accessory domains found in Dicers of higher eukaryotes but absent in Giardia Dicer. Finally, we combine the structure of Dicer with published biochemical data to propose a model for the architecture of the RNA-induced silencing complex (RISC)-loading complex.