Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

5-13-2005

Graduate Advisor

Wendy M. Olivas, Ph.D.

Committee

Shirley Bissen, Ph.D.

Teresa Thiel, Ph.D.

Marc Spingola, Ph.D.

Cynthia Dupureur, Ph.D.

Abstract

The regulation of messenger RNA (mRNA) metabolism is an important step in proper gene expression. In many eukaryotic organisms this regulation can be mediated by a group of highly conserved RNA-binding proteins known as the Puf family of proteins. The yeast Saccharomyces cerevisiae has several proteins that belong to this family. One of the yeast Puf proteins, Puf3p, binds and regulates the COX17 mRNA, which codes for a protein involved in mitochondrial copper transport. Specifically, Puf3p stimulates the decay of COX17 mRNA. However, the precise mechanism of Puf3p binding and decay regulation is yet unknown. The goal of this research is to determine the role of the Puf3p interactions required for regulation of mRNA decay in yeast, and to understand how Puf3p activity is regulated. The studies to examine Puf3p interactions have focused on the Puf3 protein sequences required for specific binding and regulation of COX17 mRNA decay. The data show that a specific region of the Puf3 protein, known as the Puf3 Repeat Domain, is sufficient to both bind COX17 mRNA and also regulate its rate of decay. Furthermore, key amino acids on the RNA-binding surface of the repeat domain that promote target specificity have been identified, as well as a specific loop structure on the protein-binding surface of the repeat domain that is required for RNA decay regulation. In addition, these studies show that the repeat domain of Puf3p directly interacts with other known mRNA decay factors, more specifically, decay factors that are involved in the deadenylation and decapping steps of mRNA decay. Additional collaborative studies have focused on the condition-specific regulation of mRNA stability in yeast. In these studies, the activity of Puf3p was found to be dependent on the available carbon source, as well as inhibited by rapamycin treatment, which in turn places the Puf3p downstream of the Target of Rapamycin (TOR) signaling pathway. Together the results from the research in this body of work will help further our understanding of transcript-specific decay mechanisms in yeast and other eukaryotes.

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Biology Commons

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