MRNA PROCESSING IN REPRODUCTION & FERTILIZATION

生殖中的 mRNA 加工

• 批准号: 6182541
• 负责人: Clinton C MacDonald
• 金额: $ 14.16万
• 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/6182541
• 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.

多聚腺苷酸化是真核生物mRNA形成其3'末端的一个必需的mRNA加工事件。 几乎所有的mRNA都是多聚腺苷酸化的，并且3'末端的形成控制着潜在的数百个基因的表达。 在雄性生殖细胞中，这些基因包括生殖细胞必需的转录因子、细胞周期调控基因、原癌基因、DNA包装基因和参与受精的基因。在体细胞组织中，mRNA多聚腺苷酸化需要信号AAUAAA。 与此形成鲜明对比的是，我们发现在雄性生殖细胞中表达的mRNA中有多达40%缺乏这种必需的多聚腺苷酸化信号。 我们提出，64,000 M的调节蛋白的切割刺激因子（CstF-64）是专门修改生殖细胞，是负责多聚腺苷酸化的mRNA缺乏AAUAAA。 我们已经证明，这种蛋白质的睾丸特异性形式在减数分裂期间特异性表达，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的基因位于X染色体上，并且在减数分裂期间失活。 因此，我们还将测试是否存在CstF-64的第二个常染色体基因，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的聚腺苷酸化模式。男性生殖细胞处理缺乏AAUAAA的前mRNA的能力在身体的所有细胞中是独一无二的，据我们所知，没有人研究这种现象。 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开始了解这个基本过程是如何在男性生殖细胞中运作的。