Examining the Conservation of Prion Formation between Yeast and Human RNA-modifying Enzymes
| dc.contributor.advisor | Garcia, David | |
| dc.contributor.advisor | Munger, Lisa | |
| dc.contributor.advisor | Shaw, Ethan | |
| dc.contributor.author | Jackson, William | |
| dc.date.accessioned | 2023-08-18T20:07:48Z | |
| dc.date.issued | 2023 | |
| dc.description | 42 pages | en_US |
| dc.description.abstract | Prions are a form of protein that can switch from their native conformation to one that can promote aggregation. This sometimes inhibits or changes their normal cellular function, causing phenotypic changes. The Garcia Lab has identified multiple RNA-modifying enzymes (RMEs) in yeast that have an inducible prion conformation, resulting in altered growth in the cells they form in. Using a CRIPSR-Cas9 mediated gene replacement, the yeast pseudouridine synthase gene PUS4 was replaced with its human orthologs TRUB1 and TRUB2 at the endogenous PUS4 locus. Potential transformant colonies were screened via PCR to ensure insertion of the human ortholog. Expression of TRUB1 and TRUB2 was validated by fusion of the human protein with a 3x HA tag to the c-terminus of the open reading frame (ORF). Western blotting showed both TRUB1 and TRUB2 are expressed in yeast. Yeast growth assays reveal that insertion of the human ortholog did not significantly reduce or impair yeast growth, suggesting that the human orthologs can functionally replace their yeast counterparts. This functional replaceability is supported by the structural similarities between the yeast and human orthologs as analyzed via protein structure visualization software. Finally, direct tRNA sequencing revealed that TRUB2, but not TRUB1, could pseudouridylate yeast tRNAs. Following transient overexpression of TRUB1 and TRUB2 and propagation for many generations, the humanized strains will be screened for paromomycin resistant growth phenotypes. It is unknown whether the human orthologs of Pus4 also have an inducible prion conformation, or if prions are responsible for any phenotypes in humans besides disease. Testing prion induction in humanized yeast is an important first step in determining if prion induction is possible in human cells, and whether prions play any role in important biological processes in humans. | en_US |
| dc.description.embargo | 9999 | |
| dc.identifier.orcid | 0000-0001-8411-0984 | |
| dc.identifier.uri | https://hdl.handle.net/1794/28748 | |
| dc.language.iso | en_US | |
| dc.publisher | University of Oregon | |
| dc.rights | CC BY-NC-ND 4.0 | |
| dc.subject | Prion | en_US |
| dc.subject | Enzyme | en_US |
| dc.subject | Conservation | en_US |
| dc.subject | Evolution | en_US |
| dc.subject | Protein structure | en_US |
| dc.title | Examining the Conservation of Prion Formation between Yeast and Human RNA-modifying Enzymes | |
| dc.type | Thesis/Dissertation |