Characterization of Missense Variants in the RNA Exosome Associated with Pontocerebellar Hypoplasia

Abstract

High-throughput sequencing has been essential in identifying genetic variants associated with disease. However, protein-level characterization of identified genetic variants is lacking, especially for rare genetic diseases. RNA sequencing and proteomics techniques were used to characterize missense variants associated with the rare neurodegenerative disorder pontocerebellar hypoplasia 1b (PCH1b). Several variants of EXOSC3, a subunit of the RNA exosome, have been identified in patients with PCH1b. We investigated three missense variants of EXOSC3, including one variant of uncertain significance (VUS). We compared the VUS with two pathogenic variants, one of which is the most common in the general population by allele frequency. The RNA exosome complex consists of 9 core subunits and 2 catalytic subunits and cooperates with several cofactors to guide its function. The RNA exosome is involved in diverse cellular functions including general RNA turnover, rRNA processing, and regulation of RNA splicing. We focused on variants in the EXOSC3 core subunit, identifying differences in protein abundance and stability changes within missense variant cell lines generated by CRISPR. While overall RNA-level changes were limited, we observed differences in rRNA processing and RNA abundance. Proteomics revealed significant decreases in RNA exosome subunit protein abundance, and interestingly, four spatially proximal subunits displayed a greater degree of reduced abundance suggesting altered interactions between / assembly of these subunits. We see potential compensation with increased protein abundance of the catalytic subunit DIS3 and several rRNA processing proteins. Comparison of the VUS with the known pathogenic variants highlights the similarities in the molecular phenotypes but determined that the effects are not as pronounced in the VUS. Ongoing experiments seek to rescue the phenotypes caused by the missense variants by proteasome inhibition and exogenous overexpression of reference or variant EXOSC3. Overall, these experiments illustrate that disease associated EXOSC3 variants have a continuum of functional changes that could explain the range of severity observed in PCH1b patients. By obtaining protein-level insights into exosome complex dynamics, we can design rescue experiments to alleviate dysfunction.