Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

12-17-2014

Graduate Advisor

Wendy M Olivas, PhD

Committee

Zolman, Bethany

Steiniger, Mindy

Kidd, Ambrose

Abstract

RNA binding proteins regulate mRNA decay and translation, two key steps in the control of gene expression in cells. Controlling mRNA metabolism allows cells to respond rapidly to altering conditions by utilizing already available mRNA, bypassing the wait for newly transcribed mRNA. The Puf family of RNA binding proteins bind specific mRNAs through interactions with sequences located in the 3’ untranslated region (UTR). Puf proteins are conserved throughout eukaryotes and have diverse roles including stem cell maintenance, neuronal development, stress response and organelle biogenesis. In Saccharomyces cerevisiae, Puf proteins are conditionally regulated in response to the cells metabolic. Specifically, in fermentative growth Puf3p is active to stimulate decay of target mRNAs; however, during respiration, Puf3p is inactive, resulting in mRNA stabilization. Presented herein, I show that in addition to Puf3p, the activity of Puf1p, Puf4p and Puf5p in yeast is conditionally regulated. I establish YHB1 mRNA as a bona fide target of Puf1p, Puf4p and Puf5p regulation through one unique binding site located in its 3’ UTR. These Puf proteins regulate YHB1 mRNA in response to nitric oxide stress. This data supports a model for Puf regulation of target mRNAs where transcripts are rapidly degraded in the absence of stress; however, in stress conditions transcripts are stabilized allowing increased protein production. I also show that Puf activity is regulated by two different mechanisms. First, the RNA binding activity of Puf5p is reduced during inactivating conditions; however, Puf1p, Puf3p and Puf4p bind target mRNAs regardless of stress. Puf proteins interact with decay machinery to facilitate mRNA degradation. The Puf3p interaction with Pop2p is disrupted in inactivating conditions and Pop2p is required to bridge interactions with other decay factors, thus preventing Puf3p stimulated decay. In a third project, I evaluated Puf proteins in humans, also called Pum proteins. I sought to identify natural targets of Pum regulation in humans. Utilizing bioinformatic and experimental approaches, I identified SNCA, LRRK2 and SAT1 as bona fide targets of Pum regulation. These mRNAs are all involved in Parkinson’s disease (PD). The implication of this discovery may provide a novel therapeutic approach to PD in the future.

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