Role of MSY2 in Male Germ Cell and Oocyte Development

MSY2 在男性生殖细胞和卵母细胞发育中的作用

• 批准号: 7049424
• 负责人: NORMAN B HECHT
• 金额: $ 34.82万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049424
• 关键词: RNA binding protein; SDS polyacrylamide gel electrophoresis; cell line; egg /ovum; embryonic stem cell; gametogenesis; gene targeting; genetically modified animals; germ cells; laboratory mouse; matrix assisted laser desorption ionization; northern blottings; phosphorylation; polymerase chain reaction; posttranscriptional RNA processing; posttranslational modifications; protein degradation; protein structure function

DESCRIPTION (provided by applicant): 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.

描述(申请人提供)：男性和女性生殖细胞都符合稳定mRNAs的需要。这一应用表明，生殖细胞特异的Y-box RNA结合蛋白MSY2对于维持雄性和雌性生殖细胞的整体mRNA稳定是必不可少的。MSY2蛋白在减数分裂和减数分裂后的雄性生殖细胞中的高水平，再加上其明显的序列独立的RNA结合活性和抑制翻译的能力，支持这一假说。同样，在发育中的卵母细胞中，MSY2蛋白的高度丰富及其在2-细胞阶段的破坏，与母体mRNAs的降解一致，支持这一假说。具体目的1将通过(1)通过两种互补的方法，即常规敲除和转基因RNAi来降低MSY2蛋白的含量，以及(2)通过转基因提高男性生殖细胞和卵母细胞中的MSY2蛋白水平，来检验MSY2是雄性生殖细胞和卵母细胞中mRNA稳定性的全局调节因子的假设。MSY2和母体mRNAs的协同降解表明MSY2周转和mRNA破坏之间存在联系。母体mRNA的周转对胚胎的持续发育是必不可少的，这表明MSY2的降解是一个关键的调节事件。越来越多的证据表明，蛋白质磷酸化的一个功能是标记磷酸化的蛋白质以供降解。《特定目的2》将通过(1)确定磷酸化位点，以及(2)评估突变这些位点对MSY2蛋白和mRNA降解的影响以及对植入前发育的功能影响，来检验这一假说，即卵母细胞中特定成熟相关的磷酸化标志MSY2的降解标志MSY2的降解，这与母体mRNA的降解在机制上是相关联的。减数分裂和减数分裂后雄性生殖细胞的高水平蛋白质合成需要许多mRNAs的持续翻译，这提出了一个问题，即在高水平的MSY2存在的情况下，这是如何发生的。具体目标3将检验这样的假设，即尽管大多数男性生殖细胞mRNAs与MSY2相关，但未处于翻译延迟之下的亚群与MSY2无关。抑制消减杂交将用于定义不受MSY2结合的mRNAs。这些实验将解决对雄配子和雌配子发育至关重要的一个重要的转录后调控机制。