Document Type

Thesis

Degree

Master of Science

Major

Biology, Molecular and Cellular Biology

Date of Defense

4-15-2005

Graduate Advisor

Wendy M. Olivas, Ph.D.

Committee

Shirley Bissen, Ph.D.

Marc Spingola, Ph.D.

Abstract

Precise regulation of gene expression is accomplished at many levels. Puf proteins are a widely conserved family of RNA binding proteins that regulate gene expression by influencing the stability of their target mRNA transcripts. Puf family members have been characterized as transcript-specific repressors in Drosophila, Dictyostelium, mouse, C. elegans, and Xenopus. In S. cerevisiae, there are 5 conserved Puf family members. Two of them, Puf3p and Puf5p, are known to destabilize their mRNA targets, down-regulating gene expression. The remaining yeast Pufs, Puf1p, Puf2p and Puf4p, are homologous to Puf3p and Puf5p, suggesting that they too have regulatory roles not yet observed. In this work, I attempt to identify new mRNA targets of Puf mediated decay in yeast. In particular, I am interested in targets of the uncharacterized yeast Pufs. The ability of a Puf protein to regulate its target transcript is dependent on Puf binding to a conserved element in the 3¿UTR of the target mRNA. A search for similar 3¿UTR elements and previous microarray data helped to identify numerous potential mRNA targets of Puf-mediated decay. In this work, experimental analysis of the candidate mRNAs positively identified three new targets of Puf mediated mRNA decay. Two of the targets, HXK1 and TIF1, are destabilized in vivo by Puf1p and Puf5p, respectively. The third target, YHB1, is actually stabilized by Puf2p. Interestingly, while the TIF1 and HXK1 3¿UTRs are sufficient for Puf mediated decay as expected, the YHB1 3¿UTR is not sufficient to confer Puf2p mediated decay onto the MFA2 coding region. In addition to these functional studies, I also demonstrate Puf interaction with conserved sequence elements in each 3¿UTR. Together, my work provides evidence that all yeast Puf proteins selectively regulate mRNA decay, and in some cases, Puf proteins may up-regulate gene expression.

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