Abstract
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.
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Abbreviations
- EJC:
-
Exon junction complex
- NMD:
-
Nonsense-mediated decay
- At:
-
Arabidopsis thaliana
- TAP tag:
-
Tandem-affinity purification tag
- GFP:
-
Green fluorescent protein
- RFP:
-
Red fluorescent protein
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Acknowledgments
We thank Michael Fromm (University of Nebraska) for providing us with the TAP-Tag expression cassette and TAP-GUS control seeds. We are grateful to Andrea Barta (Vienna Biocenter, Institute of Medical Biochemistry) for providing the SR-RFP and RFP-PRH75 clones, and Robert Mullen (University of Guelph) for providing the pRTL2ΔNS/GFP and pRTL2ΔNS/RFP clones. Mark Johnson (Brown University) and Catherine Smith (University of Calgary) provided valuable comments and assisted in editing the manuscript. We also thank Marie Fraser (University of Calgary) for assistance in the interpretation of the AtPYM binding results, and Raymond J. Turner (University of Calgary) for advice on recombinant protein expression. The Arabidopsis full-length cDNA clones were provided by the RIKEN BioResource Center. Financial support for this research was provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) award to DGM, and a Bettina Bahlsen Memorial Graduate Scholarship to NP.
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Park, Ni., Muench, D.G. Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14. Planta 225, 625–639 (2007). https://doi.org/10.1007/s00425-006-0385-y
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DOI: https://doi.org/10.1007/s00425-006-0385-y