Identification of New mRNA Targets of Puf3 Protein-Mediated Decay and Analysis of Their Condition-Specific Decay Regulation in Yeast

by Miller, Melanie A.

The eukaryotic Puf proteins function to regulate gene expression by altering mRNA stability. Specifically, Puf proteins bind the 3’ untranslated region (UTR) of mRNA targets to stimulate their turnover. In the yeast S. cerevisiae, six Puf proteins have been identified, in which Puf1p-Puf5p share a well conserved RNA-binding domain. The yeast Pufs regulate mRNA stability on a transcript-specific basis, though only a few mRNA targets have been experimentally verified. Yeast Puf3p was originally found to mediate rapid turnover of COX17 mRNA, which encodes a mitochondrial copper shuttle. More recently, microarray and computational analyses revealed that Puf3p physically associates with > 100 nuclear-transcribed mRNAs that encode mitochondrial proteins. Most of these mRNAs contain one or more conserved putative Puf3p binding site(s) within their 3’UTRs, and it is predicted that their steady-state expression levels are altered by different growth conditions. In addition, Puf3p has been shown to localize to the cytoplasmic face of the mitochondrial membrane and affect its motility, suggesting that Puf3p may play an important role in regulating mitochondrial function. In this work, I have experimentally validated several new mRNAs that are targeted for turnover by Puf3p. I have also determined that these targets are regulated by Puf3p in a condition-specific manner. Specifically, I have analyzed the decay of fifteen putative Puf3p mRNA targets in the absence and presence of Puf3p, identifying Miller, Melanie, 2007, UMSL, p.ii transcripts that represent true targets of Puf3p-mediated mRNA decay. These transcripts are rapidly degraded in the presence of Puf3p and are stabilized in a puf3? strain. Transcriptional pulse-chase experiments with one of the identified targets, CYT2, revealed that Puf3p destabilizes this target by stimulating deadenylation of the transcript and subsequent steps of decay, and this regulation only requires the CYT2 3’UTR. I have also monitored the stabilities of COX17, CYT2, and TUF1 mRNAs as reporters of Puf3p activity, and found that these RNAs are stabilized in ethanol, galactose, and raffinose conditions, suggesting that Puf3p activity is severely inhibited. Interestingly, Puf3p is rapidly activated or inactivated upon changing carbon sources to or from dextrose, respectively, as measured by CYT2 and TUF1 decay phenotypes. PUF3 mRNA and protein levels are not decreased in conditions that inhibit Puf3p activity, which suggests that Puf3p activity may be regulated post-translationally. Together, my work provides a greater understanding of the role of Puf3p in mRNA decay regulation, and provides insight into the mechanism of Puf3p activity. The significant structural and functional homology between Pufs suggests that the knowledge gained from my work on Puf3p in yeast will make contributions to understanding Puf protein regulation in higher eukaryotes as well. Miller, Melanie, 2007, UMSL, p.iii