Both male and female germ cells are confron-ted with the need to stabilize mRNAs. This application proposes that the germ cell-specific Y-box RNA-binding protein, MSY2, is essential to maintain global mRNA stability in male and female germ cells. The high level of MSY2 protein in meiotic and in post-meiotic male germ cells, coupled with its apparent sequence-independent RNA-binding activity and ability to suppress translation, supports this hypothesis. Likewise, in the growing oocyte, the very high abundance of MSY2 protein and its destruction by the 2-cell stage, coincident with the degradation of maternal mRNAs, supports this hypothesis.
Specific Aim 1 will test the hypothesis that MSY2 is a global regulator of mRNA stability in male germ cells and oocytes by (1) reducing the amount of MSY2 protein by two complementary approaches, i.e., conventional knockout and transgenic RNAi, and (2) increasing MSY2 protein levels in male germ cells and oocytes by transgenesis. The coordinate degradation of MSY2 and maternal mRNAs suggests a linkage between MSY2 turnover and mRNA destruction. The turnover of maternal mRNA is essential for continued embryo development, suggesting MSY2 degradation is a crucial regulatory event. There is a growing body of evidence that one function of protein phosphorylation is to mark the phosphorylated protein for degradation.
Specific Aim 2 will test the hypothesis that specific maturation-associated phosphorylations of MSY2 in oocytes mark MSY2 for degradation that is mechanistically linked to the degradation of maternal mRNA by (1) determining the sites of phosphorylation, and (2) assessing the effect of mutating these sites on both MSY2 protein and mRNA degradation and the functional consequences on preimplantation development. The high levels of protein synthesis in meiotic and postmeiotic male germ cells necessitates the continued translation of many mRNAs, which raises the question how this occurs in the presence of a high level of MSY2.
Specific Aim 3 will test the hypothesis that, although the majority of male germ cell mRNAs are associated with MSY2, a subpopulation not under translational delay is not associated with MSY2. Suppression subtractive hybridization will be used to define the mRNAs that are not bound by MSY2. These experiments will address an important post-transcriptional regulatory mechanism essential for both male and female gamete development.
