Research for the Cellular RNA Elongation in Mouse Fertilized Egg

生长中的卵母细胞中母体RNA的多聚腺苷酸化在非洲爪哇和小鼠中已被广泛分析。这似乎对它们的翻译很重要。最近，我们发现SSEC-D(阶段特异性表达克隆-D，现在的Bri3)在小鼠卵母细胞中表达，并被认为是受精卵受精后的多腺苷。除了SSEC-D外，Spin、-catenin、Ptp4a1和Maid在受精后表现出从头非依赖的多聚腺苷作用。为了获得受精后多聚腺苷作用的全貌，我们开发了一种新的方法来构建富含从头非依赖多聚腺苷作用的cDNA的小鼠受精卵cDNA文库。作为一项初步研究，我们在受精卵中分离到至少四个新的母体mRNAs，它们展示了Poly(A)尾巴的延伸。这是首次成功构建了富含来自受精后小鼠卵子的新的多腺化母体mRNAs的cDNA文库。接下来，我们以从文库中分离的20个具有代表性的cDNAs为探针，进行了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/或降解模式多种多样，大致可分为三种类型和七个亚型。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，而是在许多类型的母体RNA中发生的，并表明在卵子到胚胎的转变过程中，母体RNA 3‘端的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.