| Summary: | 碩士 === 臺灣大學 === 生化科學研究所 === 98 === ABSTRACT
Messenger RNA turnover plays an important role in the regulation of gene expression. Removal of the 5′ mRNA cap is an important step in both general mRNA turnover and specific mRNA decay pathways. The detailed Dcp1–Dcp2 decapping complex assembly in mammals and the regulation of decapping activity are still unclear. Compared to yeast Dcp1p, mouse Dcp1a which serves as a cofactor of decapping contains a large extra extension in the C-terminus. In this study, we aimed to explore the possible role of Dcp1a in decapping complex assembly in mammalian cells. First, we found mouse Dcp1a interacts with DDX6 and Edc3 but not Dcp2, Lsm4 or PatL1△N′. In our mapping analysis, Dcp1a interacts with DDX6 and Edc3 through its proline-rich C-terminal extension. Interestingly, when this region was deleted, the conserved EVH1 domain located in Dcp1a N-terminus could immunoprecipitate with Dcp2. On the other hand, in vivo and in vitro kinase assays confirmed the previous observation in 3T3-L1 preadipocyte early differentiation, which indicated that Dcp1a was phosphorylated by ERK signaling. The subcellular localization of Dcp1a was not affected by its phosphrylation status. In contrast, we observed Dcp1a phosphorylation appeared to activate decapping complexes by enhancing the interaction with Dcp2. Interaction between Dcp1a and other decapping regulators such as DDX6, Edc3 and Edc4 was not affected by its phosphorylation. Furthermore, two ERK-mediated phosphorylated sites at Ser 315 and Ser 319 that are located in Dcp1a C-terminus were identified by mass spectrometry analysis and demonstrated by site-direct mutagenesis combined with in vivo and in vitro kinase assay. Protein complexes immunoprecipitated by S315D/S319D phosphorylation-mimic mutant contained higher amount of Dcp2 and showed higher decapping efficiency than those by S315A/S319A mutant. Our findings suggest that the C-terminal domain of mouse Dcp1a and its post-translational modification play some roles in regulation of decapping machinery assembly.
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