The stability of Magoh and Y14 depends on their heterodimer formation and nuclear localization
Qingfeng Ma1, Takanori Tatsuno2, Yuka Nakamura2
1Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Japan; Department of Clinical Laboratory, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Abstract:
Reduced expression of the Y14 gene is a cause of Thrombocytopenia-absent radius (TAR) syndrome. This gene contains a conserved RNA recognition motif (RRM) in the central region and nuclear localization/export sequences (NLS/NES) in the N-terminal. Y14 and Magoh proteins form tight heterodimers and are the core of exon junction complexes (EJCs), which mediate various processes of mRNA metabolism after transcription. In this report, we found that protein expression levels of exogenously expressed Magoh L136R and Y14 L118R (leucine-to-arginine substitution at amino acid residue 136 and 118 respectively, that results in the formation of the complex being lost) are lower than their wild-types. This reduction is likely caused by protein levels, as no difference in mRNA levels was detected. Meanwhile, a cycloheximide chase assay determined that the degradation rates of Magoh L136R and Y14 L118R were faster than their wild-types. Both Y14 L118R and Magoh L136R lost the ability to form heterodimers with corresponding wild-type proteins. However, Y14 L118R is able to still localize in the nucleus which causes the stability of Y14 L118R to be higher than Magoh L136R. These results reveal that the stability of Magoh and Y14 is not only dependent on the heterodimer structure, but also dependent on nuclear localization.
Insights
Mutations in Y14 and Magoh proteins disrupt exon junction complexes, leading to reduced protein stability and Thrombocytopenia-absent radius (TAR) syndrome. Nuclear localization influences protein stability.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Reduced Y14 gene expression causes Thrombocytopenia-absent radius (TAR) syndrome.
- Y14 and Magoh proteins form core components of exon junction complexes (EJCs).
- EJCs are crucial for mRNA metabolism post-transcription.
Purpose of the Study:
- To investigate the impact of specific mutations (L136R in Magoh, L118R in Y14) on Magoh and Y14 protein stability and function.
- To determine the role of heterodimerization and nuclear localization in Magoh and Y14 protein stability.
Main Methods:
- Exogenous expression of wild-type and mutant Magoh and Y14 proteins.
- Quantitative analysis of protein and mRNA expression levels.
- Cycloheximide chase assay to assess protein degradation rates.
- Assessment of heterodimer formation and subcellular localization.
Main Results:
- Mutant Magoh L136R and Y14 L118R proteins showed reduced expression compared to wild-types, primarily due to increased degradation.
- Both mutants failed to form heterodimers with their wild-type counterparts.
- Y14 L118R retained nuclear localization, unlike Magoh L136R, resulting in higher stability for Y14 L118R.
- No significant difference in mRNA levels was observed between wild-type and mutant proteins.
Conclusions:
- Protein stability of Magoh and Y14 is dependent on both heterodimer formation and nuclear localization.
- Disruption of these factors contributes to the pathogenesis of TAR syndrome.
- Understanding these mechanisms provides insights into mRNA metabolism and associated genetic disorders.
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