MRNA PROCESSING IN REPRODUCTION AND FERTILIZATION

生殖和受精中的 mRNA 加工

• 批准号: 2898900
• 负责人: Clinton C MacDonald
• 金额: $ 15.37万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-27 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2898900
• 关键词: RNA binding protein; fertilization; germ cells; male; meiosis; messenger RNA; nucleic acid sequence; polyadenylate; posttranscriptional RNA processing; protein structure function; reproduction; sperm

Polyadenylation is an essential mRNA processing event by which eukaryotic mRNAs form their 3' ends. Nearly all mRNAs are polyadenylated, and 3' end formation controls expression of potentially hundreds of genes. In male germ cells these include germs cell-essential transcription factors, cell cycle regulatory genes, proto-oncogenes, DNA packaging genes and genes involved in fertilization. In somatic tissues the signal AAUAAA is required for mRNA polyadenylation. In striking contrast, we have found that as many as 40 percent of mRNAs expressed in male germ cells lack this essential polyadenylation signal. We propose that the 64,000 M regulatory protein of the cleavage stimulation factor (CstF-64) is specifically modified in germ cells and is responsible for polyadenylation of mRNAs that lack AAUAAA. We have shown that a testis-specific form of this protein is expressed specifically during meiosis, and the somatic form is absent in those cells. We propose experiments to determine the structure and function of the testis-specific CstF-64 protein. The gene for somatic CstF-64 is on the X chromosome, and is inactivated during meiosis. Therefore we will also test whether there is a second, autosomal gene for CstF-64 that is specifically expressed during and after meiosis. Finally, we will test whether the testis-specific CstF-64 protein can mediate the germ cell pattern of polyadenylation both in vitro and in vivo. The ability of male germ cells to process pre-mRNAs that lack AAUAAA is unique in all cells of the body, and, to our knowledge, no one is studying this phenomenon. Nevertheless, it has the potential to affect many of the genes that control male germ cell development and fertility. The experiments proposed here should allow us to begin to understand how this fundamental process operates in male germ cells.

多聚腺苷酸化是一种重要的 mRNA 加工事件，真核 mRNA 通过它形成其 3' 末端。 几乎所有 mRNA 都是聚腺苷酸化的，3' 末端的形成控制着数百个基因的表达。 在雄性生殖细胞中，这些基因包括生殖细胞必需转录因子、细胞周期调节基因、原癌基因、DNA 包装基因和参与受精的基因。在体细胞组织中，信号 AAUAAA 是 mRNA 多腺苷酸化所必需的。 与此形成鲜明对比的是，我们发现雄性生殖细胞中表达的 mRNA 中有多达 40% 缺乏这种必需的多腺苷酸化信号。 我们提出，裂解刺激因子 (CstF-64) 的 64,000 M 调节蛋白在生殖细胞中经过特殊修饰，负责缺乏 AAUAAA 的 mRNA 的多聚腺苷酸化。 我们已经证明，这种蛋白质的睾丸特异性形式在减数分裂过程中特异性表达，而体细胞形式在这些细胞中不存在。 我们提出实验来确定睾丸特异性 CstF-64 蛋白的结构和功能。体细胞 CstF-64 基因位于 X 染色体上，在减数分裂期间失活。 因此，我们还将测试是否存在第二个常染色体 CstF-64 基因，该基因在减数分裂期间和之后特异性表达。 最后，我们将测试睾丸特异性 CstF-64 蛋白是否可以在体外和体内介导生殖细胞多聚腺苷酸化模式。雄性生殖细胞处理缺乏 AAUAAA 的前 mRNA 的能力在身体所有细胞中是独一无二的，据我们所知，没有人在研究这一现象。 然而，它有可能影响许多控制男性生殖细胞发育和生育能力的基因。 这里提出的实验应该使我们能够开始了解这一基本过程在雄性生殖细胞中是如何运作的。