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Small RNAs in differentiating mouse ES cells

ABSTRACT: RNA-interference (RNAi) refers to a growing class of gene silencing phenomena defined by a requirement for small RNAs of 20-32 nt and the action of the Argonaute (Ago) family of ribonucleases. We have previously identified developmentally regulated small RNAs, using Northern blot analysis, that are expressed during X-chromosome inactivation in differentiating female mouse ES cels. We sought to identify these small RNAs using deep sequencing. We identified small RNAs that align to retrotransposon sequences and are enriched on the X-chromosome. LINE elements have been proposed to act as way stations during X-inactivation for the spreading of silencing along the entire chromosome, and our findings suggest that LINE elements found on the X-chromosome may be enriched for small RNAs relative to the genome. These results suggest that RNAi pathways are involved in regulating LINE elements during X-inactivation and ES cell differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE21888 | GEO | 2013/05/18

SECONDARY ACCESSION(S): PRJNA126899

REPOSITORIES: GEO

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Small RNAs in differentiating mouse ES cells

Project description:RNA-interference (RNAi) refers to a growing class of gene silencing phenomena defined by a requirement for small RNAs of 20-32 nt and the action of the Argonaute (Ago) family of ribonucleases. We have previously identified developmentally regulated small RNAs, using Northern blot analysis, that are expressed during X-chromosome inactivation in differentiating female mouse ES cels. We sought to identify these small RNAs using deep sequencing. We identified small RNAs that align to retrotransposon sequences and are enriched on the X-chromosome. LINE elements have been proposed to act as way stations during X-inactivation for the spreading of silencing along the entire chromosome, and our findings suggest that LINE elements found on the X-chromosome may be enriched for small RNAs relative to the genome. These results suggest that RNAi pathways are involved in regulating LINE elements during X-inactivation and ES cell differentiation. We size-fractionated total RNA from differentiating female mouse ES cells (day 4) into 18-24 nt and 25-45 nt populations and sequenced each fraction separately using the 454 platform. We used a karyotypically stable 40XX female ES cell line (EL16.7) with one X each of 129 and M. castaneus origins: 129 x (M.castaneus x 129).

2013-05-18 | E-GEOD-21888 | biostudies-arrayexpress

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The efficiency of Xist-mediated silencing of X-linked and autosomal genes is determined by the genomic environment

Project description:Xist is indispensable for X chromosome inactivation (XCI) in female mammalian cells. However, how Xist RNA directs chromosome-wide transcriptional inactivation of the X chromosome is largely unknown. Here, to study chromosome inactivation by Xist, we generated a system where ectopic Xist expression can be induced from several genomic contexts in aneuploid mouse ES cells. We found that ectopic Xist expression from any location on the X chromosome faithfully recapitulated endogenous XCI, showing the potency of Xist to initiate XCI. Genes that escape XCI remain consistently transcriptionally active upon ectopic XCI, regardless of their position relative to Xist transgenes, and the enrichment of CTCF at their promoters is implicated in directing XCI escape. Xist expression from autosomes facilitates their transcriptional silencing to different degrees, and gene density in proximity of the Xist transcription locus plays a central role in determining the efficiency of gene inactivation. We also show that the enrichment of LINE elements together with a specific chromatin environment facilitates Xist-mediated silencing of both X-linked and autosomal genes. These findings provide new insights into the epigenetic mechanisms that mediate XCI and identify genomic features that promote Xist-mediated chromosome-wide gene inactivation

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Project description:Higher plants encode four or five DICER-LIKE (DCL) enzymes responsible for the production of small non-coding RNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense, but in other settings, DCL2 has the opposite function of DCL4, at least in formal genetic terms. For example, knockout of DCL4 causes growth defects that are suppressed by inactivation of DCL2. Current models maintain that the biochemical basis of both of these effects is RNAi via DCL2-dependent small interfering RNAs (siRNAs). Here, we report that neither DCL2-mediated antiviral resistance nor growth defects can be explained by silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either through specific point mutations in DCL2 or simply by reducing DCL2 dosage in plants heterozygous for dcl2 knockout alleles. Intriguingly, however, all functions of DCL2 depend on it having some level of catalytic activity. We discuss this requirement for catalytic activity, but not for the resulting siRNAs, in the light of recent findings that reveal a function of DCL2 in activation of innate immunity in response to cytoplasmic double-stranded RNA.

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Antiviral RNA interference in mammalian cells

Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals. Infection of wild-type or mutant mouse ES cells and analysis of small RNAs from total extracts or immunoprecipitated components of the RNAi pathway

2013-10-10 | E-GEOD-43153 | biostudies-arrayexpress

Antiviral RNA interference in mammalian cells

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Argonaute and Trimmer generate Dicer-independent priRNAs and nucleate RNAi

Project description:RNA interference is involved in silencing of transposable and repetitive elements. How these elements are initially recognized by RNAi is a fundamental and unanswered question. We previously identified a class of Dicer-independent small RNAs, called primal small RNAs (priRNAs), in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not clear for any species. Here we reconstitute priRNA biogenesis in vitro and demonstrate that priRNAs can nucleate RNAi in vivo. We identify 3'-5' exonuclease Trimmer and show that Argonaute, loaded with longer RNA precursors, recruits Trimmer to generate the new 3' end. Next, we show that antisense priRNAs accumulate in rrp6Δ cells and nucleate RNAi at subset of protein coding genes in a Trimmer- and priRNA-dependent manner. Thus, Rrp6-mediated degradation of antisense transcripts and priRNA precursors protects the genome from spurious RNAi. Our results suggest that Argonaute association with random RNA degradation products triggers RNAi in a process of transcriptome surveillance.

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2012-04-03 | E-GEOD-32294 | biostudies-arrayexpress

Argonaute and Trimmer generate Dicer-independent priRNAs and nucleate RNAi

Project description:RNA interference is involved in silencing of transposable and repetitive elements. How these elements are initially recognized by RNAi is a fundamental and unanswered question. We previously identified a class of Dicer-independent small RNAs, called primal small RNAs (priRNAs), in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not clear for any species. Here we reconstitute priRNA biogenesis in vitro and demonstrate that priRNAs can nucleate RNAi in vivo. We identify 3'-5' exonuclease Trimmer and show that Argonaute, loaded with longer RNA precursors, recruits Trimmer to generate the new 3' end. Next, we show that antisense priRNAs accumulate in rrp6M-NM-^T cells and nucleate RNAi at subset of protein coding genes in a Trimmer- and priRNA-dependent manner. Thus, Rrp6-mediated degradation of antisense transcripts and priRNA precursors protects the genome from spurious RNAi. Our results suggest that Argonaute association with random RNA degradation products triggers RNAi in a process of transcriptome surveillance. small RNA profiling in wild type S. pombe cells and in mutant cells

2014-04-21 | E-GEOD-38636 | biostudies-arrayexpress

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