mRNA decay is a critical determinant of gene expression. Loss of mRNA stability regulation results in a variety of diseases including neurological disorders and various types of cancer. mRNA decay pathways and decay factors are conserved from yeast to humans. Our long term goalis to understand the mechanism of eukaryotic mRNA turnover using the budding yeast S. cerevisiae as the model system. A major mRNA decay pathway in yeast begins with deadenylation of the mRNA followed by decapping and 5' to 3' exonucleolytic degradation of the message. Decapping is a rate limiting step affected by several factors. The highly conserved heptameric Lsm1p-7p complex and its interacting partners, Patlp and Dhhlp, activate decapping by an unknown mechanism. This complex also protects mRNA 3'-ends from a 3' trimming reaction in vivo. It colocalizes with other decay factors and the mRNAs targeted for decay to the cytoplasmic P-bodies where decapping is thought to occur. In vivo, the Lsm1p-7p complex specifically binds to deadenylated messenger ribonucleoprotein (mRNP) molecules that are targeted for decay and are distinct from the translating mRNPs - these mRNPs are also bound to other decay factors but notto translation factors. These observations led to the following hypothesis: In a process aided by Patlp and Dhhlp, the Lsm1p-7p complex directly binds to the mRNA 3'-end after deadenylation (so that the 3'-end is protected from trimming) and triggers an mRNP rearrangement that directs the mRNP from translation to decapping. Using a combination of in vivo and in vitro studies, this proposal will test this model byfocusing on the following specific aims: (i) To elucidate the molecular events that constitute decapping in vivo. We will determine the exact basis of the block to decay in our collection of decay-defective Ism1mutants by determining how the interactions (with other decay factors and mRNA) and the localization (to the P-bodies) of the Lsm1p-7p complex are affected in these mutants, (ii)To elucidate the determinants of the Lsmlp- 7p complex-mRNA interaction. To this end, we will determine the factors that influence this interaction in vivo and the intrinsic RNA binding characteristics of the purified Lsm1p-7p complex in vitro, (iii)To determine the mechanism(s) by which the Lsm1p-7p complex enhances decapping. In principle, the Lsm1p-7p complex could enhance decapping rates in vivo either by directly stimulating the decapping enzyme or by facilitating the disassembly of the translation factors from the mRNA. We will determine the contribution of these two mechanisms to decapping activation. Together, these studies will provide insight into the mechanism of decapping and reveal the manner in which functions of the various decay factors are coordinated. Owing to the homology of the Lsm1p-7p complex to the other Sm-like protein complexes in biology, these studies should also reveal some principles common to this family of protein complexes engaged in various RNA-related functions.
