Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14.
The exon junction complex (EJC) plays an important role in post-transcriptional control of gene expression. Mago nashi (Mago) and Y14 are core EJC proteins that operate as a functional unit in animal cells, and the Mago-Y14 heterodimer interacts with other EJC core and peripheral proteins. Little is known about the biochemical and cellular characteristics of the EJC and its orthologs in plants. Here, we demonstrate that Arabidopsis Mago and Y14 form a ternary complex with PYM, an RNA-binding protein that was previously shown to interact with the Mago-Y14 heterodimer in Drosophila. Fluorescence microscopy indicated that Arabidopsis Mago and Y14 are localized primarily in the nucleus, whereas PYM is mostly cytoplasmic. In vitro pull-down assays using recombinant proteins showed that the amino-terminal region of the Arabidopsis PYM interacts with the Mago-Y14 heterodimer, a similar observation to that previously reported for the animal versions of these proteins. However, we demonstrated also that Arabidopsis PYM has the ability to interact with monomeric Mago and monomeric Y14. Immunoprecipitation and tandem affinity purification from whole cell extracts detected a subtle interaction between the Arabidopsis Mago-Y14 heterodimer and PYM in flowers, indicating that the ternary complex is not abundant in plant cells. The regions of the polypeptide responsible for nuclear import and export were defined using protein truncations and site-directed mutagenesis. This study identifies unique characteristics of Arabidopsis Mago, Y14 and PYM compared to those observed in animal cells. These are predicted to have important functional implications associated with post-transcriptional regulation of gene expression in plant cells.
Park
Nam-il
NI
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, CanadaT2N 1N4.
Muench
Douglas G
DG
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D029681:Arabidopsis Proteins; D029721:Drosophila Proteins; C569920:Mago protein, Arabidopsis; C107372:Mago protein, Drosophila; D009687:Nuclear Proteins; C569922:PYM protein, Arabidopsis; C493333:PYM protein, Drosophila; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C569921:Y14 protein, Arabidopsis; D005982:Glutathione Transferase