A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons.

Derrick J Morton,Isaac Kremsky,Brianna Brown,Kenneth H Moberg,J Christopher Rounds,Thalia Le,Lily Kim,Anita H Corbett,Maya C Karkare,Binta Jalloh,Rishi J Nair,Sara W Leung,Shaoyi Sheng,Maria C Sterrett,Khuong B Nguyen,Kathryn D Mcgaughey,Milo B Fasken,Hugo J Bellen

doi:10.1371/journal.pgen.1008901

Abstract

The 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 age-dependent 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.

Highlights

Precise production of both coding and non-coding RNAs requires regulation at the level of transcription and at the post-transcriptional level to ensure precise processing and regulated turnover [1,2,3]
Pontocerebellar Hypoplasia Type 1b (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
To elucidate the functional consequences of amino acid changes in EXOSC3 that cause PCH1b, we exploited well-established genetic approaches in Drosophila melanogaster that model EXOSC3 mutations found in individuals with PCH1b

Summary

Introduction

Precise production of both coding and non-coding RNAs requires regulation at the level of transcription and at the post-transcriptional level to ensure precise processing and regulated turnover [1,2,3]. Mutations in genes encoding structural subunits of the ubiquitously expressed RNA exosome, a critical mediator of both RNA processing and decay, cause a variety of tissue-specific diseases [3, 5, 6]. The evolutionarily conserved RNA exosome complex, first identified in Saccharomyces cerevisiae [1, 2, 7], plays critical roles in RNA processing, decay and surveillance. The multi-subunit structure of the RNA exosome complex, which is conserved from archaea to humans, consists of ten subunits [1, 2, 8, 9]. In S. cerevisiae and Drosophila, most RNA exosome subunits are termed Rrp proteins after the original yeast genetic screen for ribosomal RNA processing (rrp) mutants [7]. Studies in mice have been limited to assessing the role of Exosc in B-cells ex vivo [14]

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