Vts1 is a member of the Smaug protein family, a group of sequence-specific RNA-binding proteins that regulate mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Using RNA reporters that recapitulate Vts1-mediated decay in vivo as well as endogenous mRNA transcripts, I show that Vts1 regulates the degradation of target mRNAs in Saccharomyces cerevisiae. In Chapter Two, I focus on the mechanism of Vts1-mediated mRNA decay. I demonstrate that Vts1 initiates mRNA degradation through deadenylation mediated by the Ccr4-Pop2-Not deadenylase complex. I also show that Vts1 interacts with the Ccr4-Pop2-Not deadenylase complex suggesting that Vts1 recruits the deadenylase machinery to target mRNAs, resulting in transcript decay. Following poly(A) tail removal, Vts1 target transcripts are decapped and subsequently degraded by the 5’-to-3’ exonuclease Xrn1. Taken together these data suggest a mechanism of mRNA degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs. Previous work in Drosophila melanogaster demonstrated that Smg interacts with the Ccr4-Pop2-Not complex to regulate mRNA stability, suggesting Smaug family members employ a conserved mechanism of mRNA decay.
In Drosophila, Smg also regulates mRNA translation through a separate mechanism involving the eIF4E-binding protein Cup. In Chapter Three, I identify the eIF4E-associated protein Eap1 as a component of Vts1-mediated mRNA decay in yeast. Interestingly Cup and Eap1 share no significant homology outside of the seven amino acid eIF4E-binding motif. In eap1 cells mRNAs accumulate as deadenylated capped species, suggesting that Eap1 stimulates mRNA decapping. I demonstrate that the Eap1 eIF4E-binding motif is required for efficient degradation of Vts1 target mRNAs and that this motif enables Eap1 to mediate an interaction between Vts1 and eIF4E. Together these data suggest Vts1 influences multiple steps in the mRNA decay pathway through interactions with the Ccr4-Pop2-Not deadenylase and the decapping activator Eap1.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19085 |
| Date | 23 February 2010 |
| Creators | Rendl, Laura |
| Contributors | Smibert, Craig |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.4222 seconds