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Research for the Cellular RNA Elongation in Mouse Fertilized Egg

小鼠受精卵细胞RNA延伸的研究

基本信息

批准号：
15370005
负责人：
KIMURA Minoru
金额：
$ 8.51万
依托单位：
School of Medicine, Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Polyadenylation of maternal RNAs in growing oocytes has been extensively analyzed in Xenopus and mouse. It seems to be important for translation of them. Recently, we found that SSEC-D (stage specific expressed clone-D, now Bri3) is expressed in mouse oocytes and it is supposed to be polyadenytlated after fertilization in fertilized egg. In additon to SSEC-D, Spin, -catenin, Ptp4a1, and Maid have been found to exhibit de novo independent polyadenylation after fertilization.To obtain an overall picture of post-fertilization polyadenylation events, we developed a novel method for constructing murine fertilized egg cDNA library enriched with cDNAs exhibiting de novo independent polyadenylation. As a pilot study, we isolated at least four new maternal mRNAs exhibiting extension of poly(A) tail in fertilized 1-cell eggs. This is the first report of successful construction of a cDNA library enriched with newly polyadenylated maternal mRNAs derived from post-fertilized mouse eggs.Next, we performed RNA blot analysis to examine the elongation in maternal RNAs using 20 representative cDNA clones isolated from the library as probes. Various patterns of elongation, shortening, and/or degradation of maternal RNAs were observed from fully grown oocytes to early 2-cell embryos and could be roughly classified into three types and seven subtypes. These findings indicate that poly(A) elongation and shortening of maternal RNAs are not restricted to certain types of maternal RNAs but occur in many of them, and suggest a complex mechanism governing modification of the 3' end of maternal RNAs during the oocyte-to-embryo transition.

在非洲爪蟾和小鼠中，已经广泛地分析了生长中的卵母细胞中母体RNA的多聚腺苷酸化。这对它们的翻译来说是很重要的。最近，我们发现SSEC-D（stage specific expressed clone-D，现为Bri 3）在小鼠卵母细胞中表达，并推测其在受精卵中受精后被多聚腺苷酸化。除了SSEC-D外，Spin、β-catenin、Ptp 4a 1和Maid在受精后也表现出非从头依赖的多聚腺苷酸化，为了获得受精后多聚腺苷酸化的全貌，我们建立了一种新的方法来构建富含非从头依赖的多聚腺苷酸化cDNA的小鼠受精卵cDNA文库。作为初步研究，我们分离出至少四种新的母体mRNA，其在受精的1-细胞卵中表现出poly（A）尾的延伸。本研究首次成功构建了富含多聚腺苷酸化的小鼠受精卵母体mRNA的cDNA文库，并以该文库中分离的20个代表性cDNA克隆为探针，进行了RNA印迹分析，以检测母体RNA的延伸。从完全发育的卵母细胞到早期2-细胞胚胎，观察到母体RNA的延长、缩短和/或降解的各种模式，并可大致分为三种类型和七种亚型。这些发现表明，母体RNA的poly（A）延长和缩短并不局限于某些类型的母体RNA，而是发生在许多类型的母体RNA中，并表明在卵母细胞向胚胎过渡期间，母体RNA的3'端修饰的复杂机制。