Transcriptome profile of oocytes expressing a catalytically inactive knock-in allele of Ago2
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE57514 | GEO | 2015/01/20
SECONDARY ACCESSION(S): PRJNA246573
REPOSITORIES: GEO
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Project description:The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because miRNA function is suppressed in mouse oocytes, it has been proposed that endo-siRNAs may have a role during female meiosis. By utilizing a catalytically inactive knock-in allele of AGO2 specifically in oocytes, we disrupt the function of siRNAs and analyze the transcriptome of these oocytes in comparison with Ago2 null, and Dicer null oocytes. Germinal vesicle-intact, full-grown oocytes were collected from eCG-primed females expressing a catalytically inactive knock-in allele of Ago2 (Ago2 ADH) specifically in oocytes. Oocytes were also collected from Ago2 null and Ago2 fl/fl oocytes, as well as Dicer wild-type and null mice. Oocytes were freed of attached cumulus cells by pipetting. Twenty oocytes collected from 3 different mice were used for high-throughput RNA sequencing. Three replicates for all genotypes except Ago2 null (two). The mouse background strain is "mostly" C57BL6, i.e., it is a cross between 3 different transgenic mice of different strains.
2015-01-20 | E-GEOD-57514 | 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.
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Project description:Proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally.
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Project description:Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA as a defense against viral infection. Here, we identify 21-nt long, endogenous siRNAs (endo-siRNAs) corresponding to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to mRNAs: these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form dsRNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease, Dicer-2, and the RNAi effector protein, Ago2. We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma much as piRNAs do in the germ line. Keywords: Small RNA detection and quantification.
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Project description:MicroRNAs (miRNAs) are a large family of 19-22nt non-coding RNAs that post-transcriptionally regulate their mRNA targets. Computational algorithms predict that over half of all genes are regulated by miRNAs, yet approaches for experimental identification of miRNA binding sites are now emerging. To directly identify endogenous miRNA binding sites, we performed photo-crosslinking immunoprecipitation using antibodies against Ago2, followed by deep-sequencing of RNA tags (CLIP-seq) in mouse embryonic stem cells (mESCs). We also performed parallel CLIP-seq in Dicer null mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was mediated by miRNAs. We include the exon-array expression data obtained from three sets of Dicer WT and Dicer Null mESCs.These data are used to determine genes that are differentially expressed between Dicer WT and Dicer Null conditions. Six samples (3 Dicer wild-type CLIP RNA libraries representing two biological replicates, 2 Dicer null CLIP RNA libraries, 1 short-RNA library from Dicer wild-type mESCs) were analyzed. Six total mESC samples were analyzed (3 Dicer WT, 3 Dicer Null). Expression values for probesets were summarized into a single per-gene value. The log fold change for Dicer_WT/Dicer_Null was defined as the difference between the mean expression in Dicer WT mESCs and the mean expression in the Dicer Null mESCs.
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Project description:We aim to comprehensively characterize the small RNA population in oocytes Pseudogenes populate the mammalian genome as remnants of artifactual incorporation of coding mRNAs into transposon pathways 1. Here, we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. In these cases, endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein coding genes to antisense transcripts from homologous pseudogenes. In at least one case, an inverted repeat pseudogene gives rise to abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting in concert with piwi-interacting RNAs (piRNAs). Loss of Dicer increases expression of endo-siRNA targets, demonstrating the regulatory activity of these small RNAs. Our findings provide a function for pseudogenes in regulating gene expression via the RNAi pathway and may, in part, explain the evolutionary pressure to conserve Argonaute-mediated catalysis in mammals. Keywords: small RNAs profile
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