Characterization of Yeast Degradation Factor 1, Def1, in Post-Transcriptional Control of Gene Expression

open_access
Open Access
Author:
Cannon, Jared
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Joseph C. Reese, Thesis Supervisor
Wendy Hanna-Rose, Thesis Honors Advisor
Keywords:
Gene Regulation
RNA Degradation
Degradation Factor 1 (Def1)
Nascent Polypeptide-Associated Complex (NAC)
Egd2
Ccr4-Not Complex
Ccr4
Ribosome
Ribosome Quality Control
Transcriptional Stress
Abstract:
Gene expression fidelity depends on coordinated regulation of transcription, mRNA stability, translation, and protein degradation, and disruption of these processes can result in accumulation of defective macromolecular complexes. Degradation Factor 1 (Def1) is known to promote ubiquitin-mediated degradation of stalled RNA polymerase II (RNAPII) after DNA damage, but recent evidence suggests additional cytoplasmic roles in mRNA decay and ribosome-associated quality control. Because the Ccr4-Not complex regulates mRNA deadenylation, translation, and ubiquitination of ribosome-associated factors, and because the nascent polypeptide-associated complex (NAC) subunit Egd2 contains a ubiquitin-binding domain, this study investigated whether Def1 regulates mRNA stability through Ccr4-Not and whether Egd2 contributes to RNAPII degradation or interacts functionally with Def1. Using an MS2 tethering assay, recruitment of Def1 to a GFP reporter reduced reporter protein and mRNA abundance in wild-type cells but not in ccr4∆ cells, demonstrating that Def1-mediated repression depends on Ccr4-dependent mRNA decay. Western blot analysis confirmed that loss of repression was not due to reduced Def1-MCP expression. To determine whether Egd2 contributes to transcription-coupled degradation, Rpb1 stability was examined under transcriptional stress. Deletion of EGD2 resulted in delayed Rpb1 degradation compared to wild- type cells, although the defect was less severe than in def1∆ cells, indicating that Egd2 contributes to efficient RNAPII turnover. Growth assays demonstrated that Def1 is required for cellular fitness under stress, while depleting Egd2 did not produce a strong growth defect. Co- immunoprecipitation experiments showed that both Def1 and Egd2 associate with ribosome- associated complexes but do not detectably interact with each other under the conditions tested. Together, these findings demonstrate that Def1 promotes mRNA degradation through the Ccr4- Not complex and confirm its essential role in RNAPII degradation, while Egd2 contributes to the optimal turnover of stalled RNAPII but is not required for this process. These results suggest that Egd2 influences RNAPII degradation indirectly rather than through a direct interaction with Def1, supporting a model in which Def1 acts as a ubiquitin-dependent coordinator linking transcriptional surveillance, mRNA decay, and ribosome-associated quality control pathways.