ADrosophilaModel of Pontocerebellar Hypoplasia Reveals a Critical Role for the RNA Exosome in Neurons

Abstract

6Co-corresponding Authors Phone: 770-289-9986 (DJM) and Running title: Analysis of RNA exosome disease-linked substitutions in Drosophila EXOSC3 ortholog AbstractThe RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an agedependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction. Morton et al. 3 Author SummaryPontocerebellar Hypoplasia Type 1b (PCH1b) is a devastating genetic neurological disorder that preferentially affects specific regions of the brain. Typically, children born with PCH1b have structural defects in regions of the brain including those associated with key autonomic functions. Currently, there is no cure or treatment for the disease. PCH1b is caused by mutations in the RNA exosome component 3 (EXOSC3) gene, which encodes a structural component of the ubiquitous and essential multi-subunit RNA exosome complex.The RNA exosome is critical for both precise processing and turnover of multiple classes of RNAs. To el...

Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

“…Mutations in components of the Drosophila RNA exosome (Morton et al, 2020) It is important to note that our experiments were performed on available mutant lines from stock centers, rather than with specific patient-derived mutations. However, despite the lack of such specific mutations in our models, we observed PCH-related phenotypes in nearly all of the mutants tested.…”

The Nab2 RNA binding protein promotes sex-specific splicing ofSex lethalinDrosophilaneuronal tissue

“…At least 3 independents biological replicates per sex/genotype were tested and significance was calculated using grouped analysis on GraphPad (Prism). Lifespan was assessed at 25°C as described previously (Morton et al, 2020). In brief, newly eclosed animals were collected, separated by sex, placed in vials (10 per…”

“…Negative geotaxis was tested as previously described (Morton et al, 2020). Briefly, newly eclosed flies (day 0) were collected, divided into groups of 10 male or females, and kept in separate vials for 2-5days.…”

Vps54 Regulates Lifespan and Locomotor Behavior in Adult Drosophila melanogaster

“…However, mutations in Vps53 have been linked to the seizure phenotypes observed in pontocerebellar hypoplasia type 2E (PCH2E) suggesting that seizures may be associated with disruption of the GARP complex (Feinstein et al, 2014). Further work is required to determine if scat mutants exhibit evidence of defects in the brain like those observed in fly models for PCH (Morton et al, 2020). Finally, there is no evidence of phenotypic sexual dimorphism in the wobbler mouse.…”