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The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops [DRIP-seq]

ABSTRACT: The RNA exosome is a ten-subunit complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays many roles in regulating gene expression and protecting the genome, including modulating the accumulation of R-loops at sites of transcription. The RNA exosome interacts with specific target RNAs for decay or processing by interacting proteins termed cofactors. Attributing to the essential function of the RNA exosome for processes critical in all cells, such as production of mature rRNA, missense mutations in structural subunit genes of this complex cause neurological diseases. One possibility for why point mutations cause neurological diseases is that the RNA exosome partners with cell- or tissue-specific protein cofactors. Here, we employed a murine neuronal cell line (N2A) and performed immunoprecipitation of the RNA exosome subunit, EXOSC3, followed by mass spectrometry to obtain a snapshot of the RNA exosome interactome. We validated an interaction with DDX1, a putative RNA helicase. DDX1 plays roles in double-strand break repair, rRNA processing, and RNA metabolism. To explore shared functions of EXOSC3 and DDX1, we investigated the interaction after inducing DNA damage, and performed DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq) and RNA-seq on N2A cells depleted of either EXOSC3 or DDX1. These findings suggest that EXOSC3 and DDX1 function together in the absence of severe DNA damage to modulate spontaneous events such as RNA-DNA hybrid (R-loop) formation.

ORGANISM(S): Mus musculus

PROVIDER: GSE230115 | GEO | 2024/02/12

REPOSITORIES: GEO

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The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops [RNA-seq]

Project description:The RNA exosome is a ten-subunit complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays many roles in regulating gene expression and protecting the genome, including modulating the accumulation of R-loops at sites of transcription. The RNA exosome interacts with specific target RNAs for decay or processing by interacting proteins termed cofactors. Attributing to the essential function of the RNA exosome for processes critical in all cells, such as production of mature rRNA, missense mutations in structural subunit genes of this complex cause neurological diseases. One possibility for why point mutations cause neurological diseases is that the RNA exosome partners with cell- or tissue-specific protein cofactors. Here, we employed a murine neuronal cell line (N2A) and performed immunoprecipitation of the RNA exosome subunit, EXOSC3, followed by mass spectrometry to obtain a snapshot of the RNA exosome interactome. We validated an interaction with DDX1, a putative RNA helicase. DDX1 plays roles in double-strand break repair, rRNA processing, and RNA metabolism. To explore shared functions of EXOSC3 and DDX1, we investigated the interaction after inducing DNA damage, and performed DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq) and RNA-seq on N2A cells depleted of either EXOSC3 or DDX1. These findings suggest that EXOSC3 and DDX1 function together in the absence of severe DNA damage to modulate spontaneous events such as RNA-DNA hybrid (R-loop) formation.

2024-02-12 | GSE230120 | GEO

The Ubiquitin-Specific Protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing

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The nuclear RNA exosome is co-opted to enhance host:viral RNA hybrids that propel influenza virus ribogenesis and infectivity

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2017-05-08 | GSE96677 | GEO

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Project description:RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.

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Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

Project description:The RNA exosome is an evolutionarily conserved exoribonuclease complex that consists of a 3-subunit cap, a 6-subunit barrel-shaped core, and a catalytic base subunit. Missense mutations in genes encoding structural subunits of the RNA exosome cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. The disease symptoms vary and can manifest as neurological defects or developmental disorders. The diversity of the RNA exosomopathy pathologies suggests that the different missense mutations in structural genes result in distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models using budding yeast by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. We then performed a comparative RNA-seq analysis to assess broad transcriptomic changes in each mutant model. Three of the mutant models rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding structural subunits of the RNA exosome, EXOSC2, EXOSC3 and EXOSC5 showed the largest transcriptomic differences. Further analyses revealed shared increased transcripts enriched in translation or ribosomal RNA modification/processing pathways across the three mutant models. Studies of the impact of the mutations on translation revealed shared defects in ribosome biogenesis but distinct impacts on translation. Collectively, our results provide the first comparative analysis of several RNA exosomopathy mutant models and suggest that different RNA exosomopathy mutations result in in vivo consequences that are both unique and shared across each variant, providing more insight into the biology underlying each distinct pathology.

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2014-05-26 | E-GEOD-52539 | biostudies-arrayexpress

RNA-seq of Schizosaccharomyces pombe nuclear RNA exosome mutants

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