Characterization of the cis -acting element that targets the PPR1 mRNA for accelerated decay

The nonsense-mediated mRNA decay (NMD) pathway of the yeast Saccharomyces cerevisiae is a specialized mRNA decay pathway that targets nonsense-containing mRNAs for accelerated decay. Upf1p, Upf2p and Upf3p are required for NMD in yeast. mRNA targets of NMD are degraded rapidly in wild-type yeast cells but stabilized in upf1-, upf2- and upf3- mutants. In the first part of this dissertation we describe two improved sets of yeast strains for measuring mRNA accumulation and mRNA half-lives. The new strains have more sensitive mRNA accumulation and mRNA half-life phenotypes compared to existing strains. A number of wild-type mRNAs are also degraded by NMD. In the second part of the dissertation we show that wild-type PPR1 mRNA is one of these mRNAs. We mapped an NMD-dependent destabilizing element to a region located within the 5′ UTR and the first 92 bases of the PPR1 ORF. This element appears to target PPR1 mRNA for NMD by a novel mechanism. Ppr1p is a transcriptional activator of URA1, URA3 and possibly URA4. We used a refined Ppr1p binding consensus sequence site to search for additional genes regulated by Ppr1p. Two additional genes appear to be regulated by Ppr1p. We show that accumulation of the mRNAs for these genes is affected in a Ppr1p-dependent manner. In the third part of the dissertation, the NMD-dependent instability element from PPR1 mRNA was fused to GFP. The construct mRNA accumulates to higher levels in upf1- yeast cells than in wild-type cells, and the GFP fluorescence levels are higher in upf1- cells than wild-type cells. Thus this construct will be useful for determining the important bases within the NMD-dependent destabilizing element and a genetic screen for trans-acting factors regulating the NMD dependent stability of PPR1 mRNA