Research for the Cellular RNA Elongation in Mouse Fertilized Egg

在爪蟾和小鼠的生长卵母细胞中母体rna的聚腺苷化已经被广泛地分析。对它们的翻译似乎很重要。最近，我们发现ssc - d（分期特异性表达克隆- d，现为Bri3）在小鼠卵母细胞中表达，并在受精卵受精后被多腺苷化。除了ssc - d外，Spin、-catenin、Ptp4a1和Maid在受精后也表现出独立的聚腺苷化。为了全面了解受精后的多聚腺苷化事件，我们开发了一种新的方法来构建小鼠受精卵cDNA文库，该文库富含具有从头独立多聚腺苷化的cDNA。作为一项初步研究，我们在受精卵中分离出至少四个新的母体mrna，这些mrna表现出poly(a)尾巴的延伸。这是首次成功构建了一个cDNA文库，该文库富含来自小鼠受精卵的新聚腺苷化母体mrna。接下来，我们使用从文库中分离的20个具有代表性的cDNA克隆作为探针，进行RNA印迹分析以检测母体RNA的伸长。从完全发育的卵母细胞到早期的2细胞胚胎，母体rna的伸长、缩短和/或降解模式各不相同，大致可分为3种类型和7个亚型。这些发现表明，母体rna的poly(A)伸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，并提示在卵母细胞到胚胎的转变过程中，母体rna的3'端修饰存在复杂的机制。

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.