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The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs
1Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA.
Abstract:
In male germ cells many mRNAs are sequestered by proteins into translationally silent messenger ribo-nucleoprotein (mRNP) particles. These masked paternal mRNAs are stored and translated at specific times of germ cell development. Little is known about the mammalian testicular mRNA masking proteins bound to non-polysomal mRNAs. In this report, the major proteins binding to non-polysomal testicular mRNAs were isolated and analyzed. The two predominant proteins identified were: a Y-box protein (MSY2), the mammalian homolog to the Xenopus oocyte masking protein FRGY2/mRNP3+4, and a poly(A) binding protein. A kinase activity was also found associated with these non-polysomal RNAs. The kinase co-immunoprecipitates with MSY2 and phosphorylates MSY2 in vitro. The MSY2 associated kinase is not casein kinase 2, the kinase believed to phosphorylate mRNP3+4 in oocytes, but a yet unidentified kinase. MSY2 was found to be phosphorylated in vivo and MSY2 dephosphorylation led to a decrease in its affinity to bind RNA as judged by northwestern blotting. Therefore, testicular masked mRNAs may be regulated by the phosphorylation state of MSY2. Reconstitution experiments in which non-polysomal mRNA-binding proteins are dissociated from their RNAs and allowed to bind to exogenous mRNAs suggest that MSY2 binds RNA in a sequence-independent fashion. Furthermore, association of the non-polysomal derived proteins to exogenous non-specific mRNAs led to their translational repression in vitro.
Insights
Male germ cells store paternal mRNAs in silent particles. The Y-box protein MSY2 and a kinase regulate this process through phosphorylation, controlling RNA binding and translation.
Area of Science:
- Molecular Biology
- Reproductive Biology
- Gene Regulation
Background:
- Many mRNAs in male germ cells are stored in translationally silent messenger ribonucleoprotein (mRNP) particles.
- These masked paternal mRNAs are crucial for specific stages of germ cell development.
- The proteins involved in masking mammalian testicular mRNAs remain largely uncharacterized.
Purpose of the Study:
- To identify and analyze the major proteins that bind to non-polysomal mRNAs in mammalian testes.
- To investigate the role of protein phosphorylation in regulating mRNA masking and translation.
Main Methods:
- Isolation and analysis of proteins bound to non-polysomal testicular mRNAs.
- Co-immunoprecipitation assays to study protein interactions.
- In vitro kinase assays and Northwestern blotting to assess protein phosphorylation and RNA binding.
- Reconstitution experiments with exogenous mRNAs.
Main Results:
- The predominant proteins identified were the Y-box protein MSY2 and a poly(A) binding protein.
- A novel kinase activity associated with MSY2 was discovered, distinct from casein kinase 2.
- MSY2 phosphorylation was observed both in vitro and in vivo.
- MSY2 dephosphorylation reduced its RNA binding affinity.
- MSY2 binds RNA in a sequence-independent manner.
- MSY2 and associated proteins repressed translation of exogenous mRNAs in vitro.
Conclusions:
- Testicular masked mRNAs are likely regulated by the phosphorylation state of MSY2.
- MSY2 plays a key role in translational repression of stored paternal mRNAs.
- This study identifies a novel mechanism for post-transcriptional gene regulation in male germ cells.