A Drosophila Model of Pontocerebellar Hypoplasia Reveals a Critical Role for the RNA Exosome in Neurons

The RNA exosome is an evolutionary-conserved 3'-5' riboexonuclease complex critically important for both precise processing and 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 to understand the basis of these diseases. Mutations in the RNA exosome subunit 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b, an autosomal recessive developmental neurological disorder. The disease-causing mutations identified are missense mutations in evolutionarily-conserved regions of the protein. The tissue-specific defects these changes cause 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 begin to assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. These studies reveal a general role for Rrp40 in development of many tissues, but also highlights an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions that cause disease in humans, we developed a model of RNA exosome-linked disease in Drosophila utilizing CRISPR/Cas9 gene editing to introduce amino acid substitutions that occur in PCH1b into Rrp40. Analysis of these disease-linked amino acid substitutions in Rrp40 reveals a spectrum of behavioral and morphological phenotypes. To complement these functional studies, we performed RNA-Seq analysis to define the spectrum of RNA targets affected. Gene expression patterns show specific increases in steady-state levels of mRNAs and ncRNAs in each Rrp40 mutant consistent with impaired RNA exosome function. Furthermore, we identify several RNA exosome target RNAs that are critical for neuronal function, including Arc1, a key regulator of synaptic plasticity, that are increased in the RNA-seq dataset for both Rrp40 mutants, providing insight into why RNA exosome function is so important in neurons. 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.