Comparison of small RNA levels in Dicer helicase mutants
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE20336 | GEO | 2010/04/01
SECONDARY ACCESSION(S): PRJNA125407
REPOSITORIES: GEO
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Project description:Years after the discovery that Dicer is a key enzyme in gene-silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in C. elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently, affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not required for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wildtype and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26 nucleotide small RNA species containing a 5M-bM-^@M-^Y guanosine. We reintroduced either wildtype Dicer, or Dicer harboring a mutation (K39A) in it's helicase domain, into dcr-1(ok247) mutant worms via transgene rescue. We then used high-throughput sequencing to compare levels of small RNAs present in each of these strains.
2010-04-01 | E-GEOD-20336 | biostudies-arrayexpress
Project description:The innate immune response against viruses mainly involves type I interferon (IFN) in mammalian cells. The exact contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the type III ribonuclease DICER can generate viral siRNAs in specific conditions. In addition, it has been proposed that type I IFN and RNA silencing could be mutually exclusive responses. In order to decipher the implication of DICER during infection of human cells with the Sindbis virus, we determined its interactome by immunoprecipitation and mass spectrometry analysis. Our results show that human DICER specifically interacts with several double-stranded RNA binding proteins and helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase PKR are enriched with DICER in virus-infected cells. We validated the importance of the helicase domain of DICER in its interaction with PKR and showed that it has functional consequences for the cellular response to viral infection.
2021-04-15 | PXD019093 | Pride
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
Project description:While RNA interference (RNAi) functions as a potent antiviral innate immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double knockout human cells lacking both Dicer and PKR. Using these cells, which tolerate dsRNA expression, we show that mutant forms of human Dicer lacking the amino-terminal helicase domain can process dsRNAs to produce high levels of siRNAs that are readily detectable by Northern blot and that can effectively and specifically inhibit the expression of cognate mRNAs. However, even these more active Dicer mutants produce only modest levels of viral siRNAs in infected cells that are insufficient to inhibit viral replication. We conclude that the production of siRNAs from viral dsRNAs is likely inefficient due to the poor accessibility of viral dsRNAs to Dicer.
2015-12-02 | GSE69433 | GEO
Project description:Mammals have one Dicer gene required for biogenesis of small RNAs in microRNA (miRNA) and RNA interference (RNAi) pathways. Yet, endogenous RNAi is highly active in oocytes but not in somatic cells. Here, we provide a mechanistical explanation for high RNAi activity in mouse oocytes. The main Dicer isoform in oocytes is transcribed from an intronic MT-C retrotransposon, which functions as a promoter of an oocyte-specific Dicer isoform (denoted DicerO). DicerO lacks an N-terminal helicase domain and has a higher cleavage activity than the full-length Dicer from somatic cells. DicerO can rescue the miRNA pathway and, in addition, it efficiently produces small RNAs from long dsRNA substrates. Thus, control of endogenous RNAi activity in mice occurs via alternative Dicer isoform and the phylogenetic origin of DicerO demonstrates evolutionary plasticity of RNA silencing pathways.
2013-11-07 | GSE41207 | GEO
Project description:Mammals have one Dicer gene required for biogenesis of small RNAs in microRNA (miRNA) and RNA interference (RNAi) pathways. Yet, endogenous RNAi is highly active in oocytes but not in somatic cells. Here, we provide a mechanistical explanation for high RNAi activity in mouse oocytes. The main Dicer isoform in oocytes is transcribed from an intronic MT-C retrotransposon, which functions as a promoter of an oocyte-specific Dicer isoform (denoted DicerO). DicerO lacks an N-terminal helicase domain and has a higher cleavage activity than the full-length Dicer from somatic cells. DicerO can rescue the miRNA pathway and, in addition, it efficiently produces small RNAs from long dsRNA substrates. Thus, control of endogenous RNAi activity in mice occurs via alternative Dicer isoform and the phylogenetic origin of DicerO demonstrates evolutionary plasticity of RNA silencing pathways. NIH3T3 cells or mouse embryonic stem cells expressing oocyte-specific or somatic form of Dicer were transiently transfected with a plasmid expressing long double-stranded RNA (within the 3'-UTR of EGFP reporter) or left without transfection for controls.
2013-11-07 | E-GEOD-41207 | biostudies-arrayexpress
Project description:Dicer is an RNase III-family endoribonuclease and haploinsufficient tumor suppressor that is required for the biogenesis of miRNAs, yet in vivo structure-function characterization of its RNase IIIA and IIIB domains have not been reported. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. Inactivation of the RNase IIIA or IIIB domain blocked maturation of miRNAs derived from the 3’ or 5’ arms of miRNA precursors, respectively, and resulted in altered miRNA expression profiles. Small RNAs from murine mesenchymal stem cells (MScs) with and without Dicer (WT:Dicer f/f, KO:Dicer -/-, KO transfected with various hsDicer point mutants) were analyzed.
2012-07-13 | E-GEOD-36978 | biostudies-arrayexpress
Project description:Dicer, which is required for the processing of both microRNAs (miRNAs) and small interfering RNAs (siRNAs), is essential for oocyte maturation. Oocytes express both miRNAs and endogenous siRNAs (endo-siRNAs). To determine whether the abnormalities in Dicer knockout oocytes during meiotic maturation are secondary to the loss of endo-siRNAs and/or miRNAs, we deleted Dgcr8, which encodes a RNA binding protein specifically required for miRNA processing. In striking contrast to Dicer, Dgcr8 deficient oocytes matured normally and, when fertilized with wild-type sperm, produced healthy appearing offspring, even though miRNA levels were reduced to similar levels as Dicer deficient oocytes. Furthermore, the deletion of both maternal and zygotic Dgcr8 alleles did not impair preimplantation development including the determination of the inner cell mass (ICM) and trophectoderm. Most surprisingly, the mRNA profiles of wild-type and Dgcr8 null oocytes were essentially identical while Dicer null oocytes showed hundreds of misregulated transcripts. These findings show that miRNA function is globally suppressed during oocyte maturation and preimplantation development and that endo-siRNAs, rather than miRNAs, underlie the Dicer knockout phenotype in oocytes. We used microarrays to understand at the global level how loss of miRNAs and/or siRNAs is impacting mRNA levels in mouse oocytes.
2010-02-05 | GSE19894 | GEO
Project description:In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2, then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances. Examination of small RNAs from two different mutant strains as well as the corresponding heterozygous controls
2011-02-10 | E-GEOD-26230 | 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