The Role of Enzyme-Coactivator Interactions in the Regulation of mRNA Decapping
- Author(s): Borja, Mark
- Advisor(s): Gross, John D
- et al.
Messenger RNA degradation is a fundamental aspect of eukaryotic gene expression, regulation, and quality control. Removal of the 5' N7-methylguanosine (m7G) cap structure by the decapping enzyme Dcp2 is an irreversible step committing an mRNA to degradation by 5'-3' exonucleases. The eukaryotic decapping enzyme is composed of two subunits: Dcp2, which is the catalytic subunit, and Dcp1, and essential activator. Decapping is a tightly regulated process involving a dense network of protein-protein interactions, involving both general and pathway-specific inhibitors of the Dcp1-Dcp2 decapping complex. The purpose of this study is to characterize the interaction between the decapping complex and several coactivators of decapping at the molecular level to begin to understand how coactivators regulate the enzymatic mechanism of Dcp2. Earlier structural studies of Dcp1 and Dcp2 revealed several potential sites where coactivators could bind to the decapping complex. The crystal structure of Dcp1 reveals that it has an EVH1 fold, a protein interaction module that recognizes proline-rich sequences which bind to a surface-exposed pocket of aromatic residues. In this study we used two known enhancers of decapping in Saccharomyces cerevisiae, Edc1 and Edc2, as model coactivators to elucidate the mechanism of activation by proline-rich ligands of Dcp1. Site-directed mutagenesis, fluorescence polarization, and NMR allowed us to characterize the biochemical properties of this enzyme-coactivator system, demonstrating that Edc1 and Edc2 enhance decapping by binding the same site on Dcp1. Using a kinetic assay to monitor mRNA decapping developed in our lab, we determined the kinetic constants of the Dcp1-Dcp2 complex in the presence of coactivators Edc1 and Edc2. We found that Edc1 and Edc2 stimulate decapping by greater than 1000-fold, and that the C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step. This study is the first to demonstrate the importance of the Dcp1 proline-rich sequence (PRS) binding site in the mechanism of decapping. Our findings suggest that proline-rich ligand binding by Dcp1 is an important route utilized by the eukaryotic decapping machinery to recruit substrate and activate Dcp2.