The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops).

The RNA exosome is a ribonuclease 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 roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Analysis of RNA Exosome Subunit Transcript Abundance Across Tissues: Implications for Neurological Disease Pathogenesis.

Exosomopathies are a collection of rare diseases caused by mutations in genes that encode structural subunits of a ribonuclease complex termed the RNA exosome. The RNA exosome mediates both RNA processing and degradation of multiple classes of RNA. This complex is evolutionarily conserved and required for fundamental cellular functions, including rRNA processing. Recently, missense mutations in genes encoding structural subunits of the RNA exosome complex have been linked to a variety of distinct neurological diseases, many of them childhood neuronopathies with at least some cerebellar atrophy. Understanding how these missense mutations lead to the disparate clinical presentations that have been reported for this class of diseases necessitates investigation of how these specific changes alter cell-specific RNA exosome function. Although the RNA exosome complex is routinely referred to as ubiquitously expressed, little is known about the tissue- or cell-specific expression of the RNA exosome complex or any individual subunit. Here, we leverage publicly available RNA-sequencing data to analyze RNA exosome subunit transcript levels in healthy human tissues, focusing on those tissues that are impacted in exosomopathy patients described in clinical reports. This analysis provides evidence to support the characterization of the RNA exosome as ubiquitously expressed with transcript levels for the individual subunits that vary in different tissues. However, the cerebellar hemisphere and cerebellum have high levels of nearly all RNA exosome subunit transcripts. These findings could suggest that the cerebellum has a high requirement for RNA exosome function and potentially explain why cerebellar pathology is common in RNA exosomopathies.

The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops.

The RNA exosome is a ribonuclease 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 roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.