Characterization of factors involved in 3' to 5' mRNA degradation in yeast
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The pathways for eukaryotic mRNA degradation are composed of numerous interconnected elements. I have characterized the general roles of factors involved in 3’ to 5’ mRNA degradation pathway in the budding yeast Saccharomyces cerevisiae. The Ski2/3/8 complex [composed of a DEVH ATPase—Ski2p, a TPR (Tetratricopeptide Repeat) protein—Ski3p and a WD protein—Ski8p] and Ski7p (a putative GTPase) are essential to 3’ mRNA degradation. To better understand their role in mRNA decay, first, I explored the domain interactions within the Ski2/3/8 complex and between the Ski2/3/8 complex and Ski7p using a directed two hybrid approach combined with IP (immunoprecipitation) experiments. A model that describes all the interactions identified is presented. Since Ski2p belongs to the DEVH RNA helicase family, I then demonstrated the biochemical activity of the Ski2/3/8 complex in vitro. My results indicate that the Ski2/3/8 is a hetrotrimeric complex with the stoichiometry of 1:1:1. The complex contains ATPase activity, which is specific to Ski2p. The Ski2/3/8 complex also possesses 3’ to 5’ RNA helicase activity. The duplex unwinding activity is intrinsic to Ski2p and requires the ATPase activity of Ski2p. More importantly, the helicase acitivity of Ski2p is stimulated by the presence of a short poly(A) tail (A12). Interestingly, mutations in Ski2p changing DEVH to AEVA cause this protein to be dominant negative. The dominant negative phenotype is caused by outcompeting wildtype Ski2p for incorporation into the Ski2/3/8 complex and forming a large inactive complex with the exosome. To better understand the role of GTPase Ski7p in 3’ mRNA decay, a dominant negative screen of SKI7 was carried out. Further characterization of the identified dominant negative mutants of SKI7 revealed that these mutants altered the interaction with either the Ski2/3/8 complex or the exosome, supporting the idea that Ski7p functions as a mediator between the Ski2/3/8 complex and the exosome in 3’ to 5’ mRNA degradation.