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Requirement for RNA-binding activity of MSY2 for cytoplasmic localization and retention in mouse oocytes
Junying Yu1, Norman B Hecht, Richard M Schultz
1Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
Abstract:
MSY2, a mouse germ cell-specific Y-box protein, is implicated in the global regulation of the stability and/or translation of maternal mRNAs in the mouse oocyte. We report here that in the oocyte approximately 75% of MSY2 protein is associated with a Triton-insoluble preparation, whereas in either male germ cells or when exogenously expressed in transfected somatic cells almost all MSY2 is soluble. This retention in the oocyte, which is unlikely mediated either by microfilaments or by microtubules, markedly decreases beyond the two-cell stage of development. By microinjecting mutant MSY2-EGFP chimeric mRNAs into mouse oocytes and then assaying the expressed protein's localization by laser-scanning confocal microscopy, we find that an intact cold-shock domain (CSD), containing two RNA-binding motifs, is required to localize MSY2 to the oocyte cytoplasm. In addition, an additional basic/aromatic amino acid island (B/A), which can also interact with RNA, in the C-terminal tail domain is necessary to retain MSY2 following Triton permeabilization. Intact mRNA appeared required for this retention, since RNase A treatment of Triton-permeabilized oocytes or microinjection of RNase A into the oocyte released essentially all of the endogenous MSY2 protein. Furthermore, there is a positive correlation between the ability of the mutant MSY2-EGFP protein to remain associated with the Triton-insoluble preparations and its increased affinity for RNA, as determined by RNA electrophoretic mobility shift assays. These results suggest that binding of intact maternal mRNA by MSY2 is required for its cytoplasmic retention.
Insights
Mouse Y-box protein 2 (MSY2) is retained in the oocyte cytoplasm by binding to intact maternal mRNA. This interaction is crucial for MSY2 localization and function during early development.
Area of Science:
- Molecular and Cellular Biology
- Developmental Biology
- Reproductive Biology
Background:
- MSY2 is a germ cell-specific Y-box protein involved in maternal mRNA regulation in mouse oocytes.
- Oocyte-specific retention of MSY2 differs from its soluble state in male germ cells and transfected somatic cells.
- MSY2 retention decreases significantly after the two-cell developmental stage.
Purpose of the Study:
- To investigate the molecular mechanisms underlying MSY2 protein retention in the mouse oocyte cytoplasm.
- To identify specific protein domains and interactions required for MSY2 localization and retention.
- To determine the role of maternal mRNA binding in MSY2 cytoplasmic retention.
Main Methods:
- Microinjection of mutant MSY2-EGFP chimeric mRNAs into mouse oocytes.
- Laser-scanning confocal microscopy for assessing protein localization.
- Triton-insoluble preparation and RNase A treatment to analyze protein association.
- RNA electrophoretic mobility shift assays (EMSA) to determine RNA-binding affinity.
Main Results:
- An intact cold-shock domain (CSD) is essential for MSY2 cytoplasmic localization.
- A basic/aromatic amino acid island (B/A) in the C-terminal tail is necessary for Triton-insoluble retention.
- RNase A treatment or microinjection releases endogenous MSY2, indicating dependence on intact mRNA.
- MSY2 retention correlates positively with its affinity for RNA.
Conclusions:
- MSY2 cytoplasmic retention in oocytes is dependent on the binding of intact maternal mRNA.
- Specific domains within MSY2, including the CSD and B/A region, mediate its localization and retention.
- These findings elucidate a novel mechanism for maternal factor regulation during early embryogenesis.