Tag: David Garcia

The Role of Ribosome-Associated Protein Quality Control in a Prion-Based Epigenetic State

Presenter: Phaedra Whitty – Biochemistry

Faculty Mentor(s): David Garcia

Session: (In-Person) Poster Presentation

A prion that has been discovered in yeast, [BIG+], allows cells to adopt a ‘live fast, die young’ strategy, accelerating growth rate at the cost of a shortened lifespan. Prions are heritable, alternatively- structured proteins that are implicated in many mammalian neurodegenerative diseases. However, they can also act as a beneficial epigenetic mechanism by altering gene expression in cells. The [BIG+] prion is a form of Pus4, an RNA-modifying enzyme conserved throughout nature, and has been shown to affect protein synthesis. The epigenetic state induced by it is characterized by accelerated cell proliferation, increased cell size, shortened lifespan, and increased translation activity. The mechanism of the [BIG+] prion remains unknown. This project investigates the ribosome-associated protein quality control pathway (RQC), a cellular system to monitor issues in translation, as a potential contributor to the [BIG+] phenotype. A genetic approach was taken to knock out each of four genes coding for proteins involved in RQC. These strains were used in luciferase reporter assays to examine the difference in translation phenotypes between mutant naïve and mutant [BIG+] strains. A notable change in the [BIG+] translation phenotype of mutants as compared to wild-type strains suggested the involvement of at least two RQC proteins, Hbs1 and Rli1, in the prion phenotype. These results are some of the first mechanistic insights into how this prion affects translation.

Misfolded but not Malicious: Prion Proteins in Budding Yeast

Presenter: Mikala Capage − Biology

Faculty Mentor(s): David Garcia

(In-Person) Poster Presentation

Prion proteins, although frequently associated with neurodegenerative diseases, are not universally harmful to cells. Instead, prions may serve as a beneficial epigenetic mechanism, allowing cells
to alter their phenotype to adapt to adverse environmental conditions. Prions form when a protein adopts alternate folding conformation. The Garcia Lab aims to identify beneficial prions using the budding yeast, Saccharomyces cerevisiae. We are particularly interested in prion conformations of RNA modifying enzymes (RMEs), because these proteins can affect the expression of many genes simultaneously. After screening hundreds of yeast strains, the Garcia Lab has identified six strains of yeast—associated with the RMEs Abd1, Cet1, Ppm2, Pus4, Pus6 and Trm5—that exhibit resistance to harmful chemicals . Extensive tests are needed to confirm that the resistance to stress is caused by a prion conformation of an RNA modifying enzyme. Here, data describing the meiotic inheritance, protein dependance, and cytoplasmic inheritance are presented. Taken together, these results are key in attributing the previously identified growth states to a prion conformation of each of the six RNA modifying enzymes . The Garcia lab will continue to investigate these putative prions in future experiments to determine the mechanism for resistance. This research represents an important contribution to our understanding of prions as a protein-based epigenetic mechanism and their effects on key cell processes.

Influence of Misfolded Proteins on the Growth Pathway in Budding Yeast

Presenter(s): Zachary Basham

Faculty Mentor(s): David Garcia

Poster 3

Session: Sciences

Prions are misfolded proteins that have developed a negative connotation due to their involvement in many degenerative diseases. However, some prions have been found in yeast that result in benefits for the cell. This experiment focused on a specific prion that gives rise to larger cells with increased replication rate. We hypothesized that the prion must be interacting with a pathway that regulates the maturation of the cell. To determine the cause of this phenotype, we grew cells in the presence of rapamycin, an inhibitor of the TOR (Target of Rapamycin) complex which regulates the growth of cells by modifying proteins. By recording the absorbance of cell cultures with and without the prion, we were able to determine the growth rate and support the claim that the misfolded protein is influencing TOR because they showed resistance to the drug. The next step is to determine what is being affected in the complex to provide this result. Understanding how prions work on a molecular level may reveal new cell functions not possible by genetics alone.

Propagating Putative Prion States in RNA Modifying Proteins

Presenter(s): Jacob Evarts—Computer and Information Science

Faculty Mentor(s): David Garcia

Session: Prerecorded Poster Presentation

Prions have been closely associated with fatal neurodegenerative diseases such as mad cow disease . However, recent evidence suggests that prions provide an additional class of epigenetic mechanism that works at a rapid pace . From an evolutionary standpoint, the ability to change phenotypes without waiting for genetic variance would be hugely beneficial in a high stress environment . Using two known techniques for increasing de novo prion formation, protein overexpression and environmental stressor, we performed a large-scale screen across many RNA modifying enzymes in budding yeast . The growth dynamics presented here suggest that putative prion-state induction could be a widespread epigenetic mechanism across yeast .

Hunting for prions: Using inheritance patterns in yeast cells to attribute epigenetic states to prion proteins

Presenter(s): Mikala Capage—Biology

Faculty Mentor(s): David Garcia

Session 2: Cells R Us

The Garcia Lab studies the effects of prion proteins on key biological processes using the budding yeast, Saccharomyces cerevisiae . Prions can influence a cell’s phenotype but are based on a heritable protein confirmation and not sequence differences in the nucleic genome . Prions are inherited through the cytoplasm in a pattern of “non-Mendelian” inheritance in which all the cell’s offspring inherit the phenotype caused by the prion . To order to continue to research the broader impacts of prion proteins on biology, it is necessary to identify new examples of them. Our lab recently identified five new candidate prions–of proteins that chemically modify RNA–in yeast that exhibited heritable growth traits after exposure to chemical stressors . To test if the previously observed growth traits inherit in a pattern consistent with a prion, this project uses central methods in
yeast genetics including tetrad dissections, cytoductions, and growth assays . This project has the potential to significantly add to the list of known yeast prions, particularly those involved in RNA biology . A broader understanding of how prions function in yeast will eventually help us transition to understanding the roles they may have in human cells . This is an ongoing project; presented here will be a description of the methods, preliminary and expected results, background information for each putative prion, and other aspects of this experiment .

Influence of a prion protein on the TOR pathway in Saccharomyces Cerevisiae

Presenter(s): Zack Basham—Biochemistry

Faculty Mentor(s): David Garcia

Session: Prerecorded Poster Presentation

Pseudouridine synthases are critical RNA modifiers in eukaryotes . One member of this family of enzymes, encoded by the Pus4 gene in the budding yeast Saccharomyces cerevisiae, forms a prion protein, named [BIG+] . Rather than resulting in cell death, as for known mammalian prion diseases, [BIG+] promotes increased cell proliferation and cell size . These observations raise the question of how the prion promotes cell growth . The increased cell size and growth rate suggests an alteration to a fundamental eukaryotic growth control pathway, mediated by the TOR complex (“target of rapamycin”) . One target of TOR, a protein kinase, is Sch9, an AGC kinase, which is activated via phosphorylation by the TOR complex . Sch9 is involved in multiple processes essential for growth such as ribosome biogenesis, translation control, and cAPK activity . To better understand the relationship between [BIG+] and TOR, we have introduced hyperactive mutants of TOR or Sch9 into [BIG+] and naive cells . By monitoring growth rate in media with varying levels of arginine, we can monitor [BIG+] response to different nutrient conditions . We have also made progress in monitoring the expression of Sch9 in [BIG+] cells compared to cells without the prion . This contributes to our understanding of how the prion and TOR complex are interacting to affect cell growth .