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Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype D]

ABSTRACT: The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1Δ mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans.

ORGANISM(S): Cryptococcus neoformans var. neoformans JEC21

PROVIDER: GSE21177 | GEO | 2010/05/01

SECONDARY ACCESSION(S): PRJNA129663

REPOSITORIES: GEO

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Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype D]

Project description:The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1Δ mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans. RNAi_D: 6 slides are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted from 4 strains (NE519 MAT alpha, NE520 MAT a backcrossed Wild type strain, NE517 rdp1 mutant MAT alpha, NE518 rdp1 mutant MAT a). We use Cy5 as rdp1 mutant dye and Cy3 as a WT dye

2010-05-01 | E-GEOD-21177 | biostudies-arrayexpress

Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans

Project description:The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1Δ mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans. This SuperSeries is composed of the SubSeries listed below.

2010-05-01 | GSE21178 | GEO

Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype A]

2010-05-01 | GSE21176 | GEO

Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans

Project description:The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1? mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans. This SuperSeries is composed of the following subset Series: GSE21176: Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype A] GSE21177: Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype D] Refer to individual Series

2010-05-01 | E-GEOD-21178 | biostudies-arrayexpress

Molecular and Functional Characterization of the role of RNA silencing components in Cryptococcus neoformans [RNAi_Serotype A]

Project description:The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1Δ mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans. RNAi_A: 16 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 3 strains (H99 Wild type strain, ago1Δ , rdp1Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. 2nd experiment sample are dye swaped.

2010-05-01 | E-GEOD-21176 | biostudies-arrayexpress

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1∆, ago1∆, or rdp1∆ cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin.

2013-12-26 | GSE52444 | GEO

A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeast C. neoformans

Project description:The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. The RDE genes impact global siRNA levels and transposon mobilization but not global transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies reveal that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.

2020-07-13 | GSE128009 | GEO

The impact of RNAi on the Saccharomyces cerevisiae transcriptome

Project description:The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. Here, we show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial dsRNA virus, known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species, in that RNAi is absent in all species known to possess dsRNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution. Examine mRNA abundance of S. cerevisiae wild-type (DPB249), +AGO1 (DPB252), +DCR1 (DPB255) and +AGO1, DCR1 (DPB258).

2011-08-10 | E-GEOD-31288 | biostudies-arrayexpress

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1M-bM-^HM-^F, ago1M-bM-^HM-^F, or rdp1M-bM-^HM-^F cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin. Examination of genome-wide enrichment in H3K9 dimethylation in fission yeast haploid cells

2013-12-26 | E-GEOD-52444 | biostudies-arrayexpress

An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer

Project description:Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleo-cytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

2011-09-23 | GSE30586 | GEO

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