Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

7-17-2009

Graduate Advisor

Wendy M. Olivas, Ph.D.

Committee

Cindy Dupureur, Ph.D.

Marc Spingola, Ph.D.

Bethany Zolman, Ph.D.

Abstract

The stability of a messenger RNA (mRNA) is a highly regulated and important aspect of gene expression. Proteins that regulate mRNA stability often bind to 3' untranslated region (UTR) sequence elements. The eukaryotic Puf proteins are one class of 3'UTR binding proteins that regulate the stability and expression of their target transcripts. Several global genome analyses have identified hundreds of potential mRNA targets of the Saccharomyces cerevisiae Puf proteins, however only three mRNA targets for these proteins have been characterized thus far. After direct testing of nearly forty candidate mRNAs, I have established three of these as true mRNA targets of Puf-mediated decay in yeast, YHB1, HXK1 and TIF1. In a novel finding, multiple Puf proteins, including Puf1p, regulate HXK1 and TIF1 mRNAs in combination. TIF1 mRNA decay can be stimulated individually by Puf1p and Puf5p, but the combination of both proteins is required for full regulation. This Puf-mediated decay requires the presence of two UGUA binding sites within the TIF1 3'UTR, with one site regulated by Puf5p and the other by both Puf1p and Puf5p. The stability of the endogenously transcribed HXK1 mRNA, cellular levels of Hxk1 protein activity, and HXK1 3'UTR-directed decay are affected by Puf1p and Puf5p as well as Puf4p. YHB1 mRNA decay is mediated by Puf5p and also requires a UGUA sequence element. This work has discovered the first targets of Puf1-mediated decay. Since much of our knowledge of the mechanism suggests that Puf protein target recognition and mechanism of action varies with each of these proteins, I investigate the mechanism of Puf1p-mediated decay using a variety a techniques. The results of this research aid in our understanding of the similar, yet distinct, decay regulation of Puf proteins in yeast and higher organisms.

Included in

Biology Commons

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