Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease caused by CAG repeat expansion in the ATXN3 gene, which results in polyglutamine expansion in the ataxin-3 protein. Ataxin-3 is a deubiquitinase and is critically involved in proteostasis, yet how polyglutamine expansion in ataxin-3 alters its deubiquitinase function and contributes to SCA3 pathogenesis remains unclear. To identify proteins that may be differently deubiquitinated by polyglutamine-expanded ataxin-3 and thus important to SCA3 pathogenesis, we immunoprecipitated K48-ubiquitinated proteins in Neuro-2A cells expressing wild-type or polyglutamine-expanded human ataxin-3, or no human ataxin-3 and performed LC-MS/MS analysis. ‘RNA metabolism proteins’ was the largest class of dysregulated proteins in cells expressing polyglutamine-expanded compared to wild-type ataxin-3, including a large number of RNA-binding proteins implicated in amyotrophic lateral sclerosis (ALS), a related neurodegenerative disease. We subsequently discovered that the ALS-linked RNA-binding protein matrin-3 is increased in abundance in Neuro-2A cells expressing wild-type human ataxin-3, compared to deubiquitinase inactive ataxin-3, suggesting an important role of ataxin-3’s deubiquitinase function in regulating matrin-3 proteostasis. We also identified an increased abundance of matrin-3 protein in the brain of SCA3 mice, and determined by immunofluorescent staining and co-immunoprecipitations that ataxin-3 and matrin-3 proteins co-interact in cells. Our findings highlight a role for ataxin-3’s deubiquitinase function in regulating the proteostasis of the ALS-linked RNA-binding protein matrin-3, which may be important in SCA3 and ALS pathogenesis. We are continuing to investigate how ataxin-3’s deubiquitinase function can be targeted to regulate the proteostasis of RNA-binding proteins in neurodegenerative diseases including ALS and SCA3.