| The exon junction complex (EJC) plays an important role in the post-transcriptional control of gene expression. Mago nashi (Mago) and Y14 are core EJC proteins that operate as a functional unit in animal cells to regulate the nuclear export, localization, translation, and quality of mRNAs. The Mago-Y14 heterodimer interacts with other EJC core and peripheral proteins, such as PYM. This thesis was aimed at providing insight into the role that Arabidopsis (At) Mago, AtY14 and AtPYM plat in the biochemical, cellular and developmental biology of plants.; In vitro pulldown assays using recombinant proteins demonstrated that AtMago, AtY14, and AtPYM interact. Unlike the animal orthologs of these proteins, AtPYM was shown to bind with monomeric AtMago and monomeric AtY14. The regions of the polypeptide responsible for nuclear import and export were defined using transient expression of truncated proteins fused to fluorescent proteins, and site-directed mutagenesis. The RNA binding activity of AtPYM was also demonstrated. Fluorescence microscopy indicated that AtMago and AtY14 are primarily localized to the nucleus, whereas AtPYM is mostly cytoplasmic but shuttles between these two compartments.; Immunoprecipitation and tandem affinity purification from whole cell extracts detected a subtle interaction between AtY14 and AtPYM, indicating that there is at least a subtle interaction between these proteins in vivo. Immunoprecipitation was used to identify several putative AtY14 and AtPYM interacting proteins using LC-MS/MS analysis. Selected proteins from those that were identified had their interactions analyzed in greater detail.; Plants with reduced levels of AtMago expression by RNA interference (RNAi) have developmental phenotypes that included a disorganized meristem, fused stems, abnormal flowers, and abnormal pollen development. Reverse transcription (RT)-PCR analysis showed that AtMago and AtPYM do not have an obvious role in nonsense-mediated mRNA decay (NMD). This suggests that the plant EJC is not involved in NMD in plants. In situ hybridization of profilin mRNAs in root hair cells demonstrated that RNAi- AtMago knockdown affects the localization of this transcript to the root hair tip, indicating a role for AtMago in mRNA localization. |
