Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology

Date of Defense

9-17-2010

Graduate Advisor

Wendy M. Olivas, Ph.D.

Committee

Spingola Marc

Thiel Teresa

Dupureur Cynthia

Abstract

Proper regulation of gene expression at a cellular level is required in all organisms for their successful adaptation and survival to physiological or environmental changes. In eukaryotes, a convenient way of regulating gene expression is achieved by post-transcriptionally adjusting the decay rates of different mRNAs. The Puf family of proteins in yeast belong to a widespread group of eukaryotic RNA-binding proteins that regulate the lifespans of target mRNAs by sequence specifically binding to 3' untranslated regions (UTRs) and modulating their decay rates. For example, the yeast Puf3 protein binds the COX17 3' UTR, stimulating its deadenylation and subsequent decay. However, the specific mechanism by which Puf3p regulates these decay processes was not known. In this research, insight was gained on Puf3 protein interactions and its mechanism of action for COX17 mRNA regulation. Through biochemical and genetic approaches, several decay factors involved in decapping and deadenylation events were identified to bind Puf3p via protein-protein interactions. Specifically, a four amino acid loop structure on the outer surface of Puf3p (R7A loop) was found to be the interaction point to which Pop2p directly (and Dhh1p indirectly through Pop2p) binds the Puf3RD. Other decay factors were found to bind Puf3RD independent of the R7A loop and Pop2p. Puf3p activity was also analyzed under different environmental conditions. Finally, four new Puf4p mRNA targets (Rrs1, YJL122W, Ebp2 and Pus7) were experimentally determined. All of these Puf4p target RNAs were also regulated by Puf5p, suggesting that combinatorial regulation of RNAs by Puf4p and Puf5p is a common mechanism. In conclusion, the results from this research provide insight into the mechanism of Puf protein action and contribute to the understanding of Puf3p interactions that function to regulate mRNA decay in yeast. In addition, this research provides evidence that physiological conditions play a key role in post-translational regulation of Puf3p activity and therefore mRNA decay. Given the structural and functional similarities between Puf proteins, these results will significantly increase our understanding of the role of Puf proteins in yeast and other eukaryotes.

OCLC Number

669839125

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