Ataxin-2 is an RNA-binding protein that is involved in many crucial cellular processes such as R-loop regulation, mRNA stability, TOR signaling regulation, and stress granule formation. Ataxin-2 is highly conserved, found in organisms ranging from Saccharomyces cerevisiae to Caenorhabditis elegans and Homo sapiens. Recently, ataxin-2 has been linked to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal disease that causes loss of motor neurons. In addition to ataxin-2 interacting with known ALS risk factor proteins, research into the relationship between ataxin-2 and ALS shows that polyglutamine expansions in ataxin-2 are gain-of-function mutations that lead to overactivity of ataxin-2 and probable neurodegeneration. In fact, targeting ataxin-2 using gene silencing techniques dramatically slows the progression of ALS in both mice and man.The Grose laboratory has characterized a serine-threonine kinase, PAS kinase as upstream kinase and putative activator of ataxin-2. We hypothesize that knockdown of PAS kinase could, therefore, have similar effects to directly downregulating ataxin-2 and its cellular functions. Characterization of Ataxin-2 has revealed that its gain or loss of function lead to distinct cellular phenotypes. One study concluded that lowering ataxin-2 levels reduced the size and number of stress granules in mammalian cells, which was observed through microscopy. Another study found that activation and overexpression of ataxin-2 lead to reduced mTOR levels because of its sequestration to stress granules. Lastly, preliminary data obtained by the Grose laboratory suggests that yeast deficient in Pbp1 (the yeast homologue of ataxin-2) have altered cell cycles.This project describes the cellular readouts used to determine if PAS kinase downregulation confers the same cellular phenotypes as ataxin-2 downregulation. First, we found that PAS kinase does influence ataxin-2 abundance in mammalian cells. Using yeast as a model, we found that Pbp1 influences the cell cycle through its binding partners, causing a reduction in the percentage of cells in the G2 phase compared to the G1 phase. PAS kinase conferred an opposite change, most likely due to the activity of other PAS kinase substrates. Additionally, we found that Pbp1 deficiency is synthetically lethal when in conjunction with deficiency of any one of its cell cycle-related binding partners. The cellular changes cause by Pbp1 deficiency highlight not only the importance of ataxin-2 in the cell, but also the importance of understanding the effects of downregulation of ataxin-2.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-9574 |
| Date | 01 July 2019 |
| Creators | Melhado, Elise Spencer |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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