PROJECT SUMMARY: The RNA exosome, an essential molecular machine that contributes to processing and/or decay of nearly every species of RNA, is a multi-subunit complex that is conserved across all eukaryotes in both sequence and structure. Recently, mutations have been identified in genes that encode structural subunits of the RNA exosome. Although these mutations all occur in genes that encode components of the same complex, they cause distinct, tissue specific human disease, including neurodegenerative diseases and developmental disorders. This growing collection of RNA exosome-linked diseases can be classified as “exosomopathies”. The disease- causing mutations are missense mutations that alter single amino acids in conserved regions of these structural subunits. Explaining the tissue-specific nature of these diseases is challenging if the amino acid substitutions generally affect the molecular function(s) of this complex. Rather, the different amino acid substitutions could have distinct consequences that differentially affect key interactions and/or the integrity of the complex, ultimately disrupting RNA targeting/processing. I hypothesize that distinct disease-causing amino acid substitutions differentially impact the function of the RNA exosome. The studies proposed here will compare the in vivo consequences of two exosomopathy mutations identified in the structural subunit genes EXOSC2 and EXOSC5 (identified by our clinical collaborator), using Saccharomyces cerevisiae. Mutations in EXOSC5 are linked to cerebellar degeneration, while mutations in EXOSC2 are linked to a novel syndrome characterized by retinitis pigmentosa, hearing loss, premature aging and mild intellectual disability. We have already generated S. cerevisiae models for each of these disease-linked amino acid substitutions: EXOSC2 amino acid substitution, G226D in the yeast orthologue Rrp4, and the disease-linked EXOSC5 amino acid substitution, L191H in the yeast orthologue Rrp45. Each of these amino acid substitutions causes a temperature sensitive growth defect that can be exploited in yeast genetics approaches. Importantly, my preliminary data reveal that these mutations are differentially suppressed by overexpression of distinct RNA exosome cofactors, factors that interact with the complex to confer target specificity. These preliminary data suggest distinct in vivo consequences for these two mutations, illustrating the importance and value of studying the molecular underpinnings of each exosomopathy mutation in an in vivo system. To achieve this goal, I will 1) examine RNA exosome complex integrity comparing the two exosomopathy mutant models (Aim 1); 2) assess differentially affected RNA exosome interactions in exosomopathy mutant models using both targeted biochemical assays and discovery-based genetic screens (Aim 2); and 3) employ RNA-Seq to define the spectrum of RNAs altered by exosomopathy-modeled amino acid substitutions (Aim 3). This proposed comparative study will reveal how different amino acid substitutions in structural subunits of the RNA exosome impair the function of this essential complex, providing insight into how different exosomopathy mutations could cause distinct clinical manifestations.

PROJECT NARRATIVE: The mRNA content of a given cell, which dictates the form and function of that cell, is regulated in part by evolutionarily conserved RNA decay machines such as the RNA exosome. Recent clinical reports identify mutations in genes encoding subunits of the RNA exosome complex that cause human disease, with distinct clinical manifestations. Here, I employ a genetic model system to understand how the mutations identified in patients alter the function of the essential RNA exosome complex, which will provide insight into the distinct symptoms seen in patients.