Analysis of localization mechanisms of postplasmic RNAs in early ascidian embryos.

我们已经从海鞘卵中分离出了许多rna，这些rna被称为花叶卵，其中一些rna定位于卵后区域，这些rna被认为是胚胎发生过程中轴规范和不平等切割模式所必需的，因此被称为质后rna。本研究的目的是通过鉴定控制其定位的顺式元件和与这些元件结合的反式作用因子，阐明亚洲卵和胚胎中RNA分选的分子机制。通过分析上述六种RNA的详细分布模式，我们发现它们根据定位途径可分为两类；I型和II型。注射标记的后质rna合成rna表明，它们的31个UTR序列对于胚胎中rna的正确定位是必要和充分的，因为这两种类型的rna。此外，我们已经将这些区域最小化到约50 nt。为了识别和表征与这些区域结合的反式作用因子，我们正在进行凝胶转移实验、东西方分析和使用顺式元件的UV交联分析，通过这些实验，我们正在建立检测系统，以寻找卵子中序列特异性rna结合蛋白。同时，rna结合蛋白的生化纯化和编码蛋白的cdna的分离也在进行中。另一方面，通过电镜原位杂交分析，我们发现这两种类型的质后rna定位于质后的超结构不同部位。这将使我们对海鞘胚胎发生过程中RNA的定位机制有更系统的认识。

We have isolated many RNAs localized in ascidian eggs, which are known as a mosaic egg, Among them, a number of RNAs are localized in the posterior region that are suggested to be essential to the axis specification and unequal cleavage patterns during embryogenesis, and are therefor named postplasmic RNAs. The aim of this study is to clarify the molecular mechanisms of the RNA sorting in asicdian eggs and embryos through identification of cis-elements controlling their localization and of trans-acting factors that bind to the elements. By analysis of the detailed RNA distribution patterns of six of the above-mentioned RNAs, we found that they are categorized into two groups by the localization pathways ; type I and type II. Injection of labeled synthetic RNAs of the postplasmic RNAs revealed that their 31 UTR sequences are necessary and sufficient for the correct localization of the RNAs in the embryos as th the both types of the RNAs. Furthermore, we have minimized the regions to about 50 nt. In order to identify and characterization of the trans-acting factors that bind to the regions, gel-shift experiments, East-Western analysis and UV cross-linking analysis using the cis-elements are on-going, by which we are establishing the assay systems to find sequence-specific RNA-binding proteins in the egg. Simultaneously biochemical purification of the RNA-binding proteins and isolation of the cDNAs encoding the proteins are also in progress. On the other hand, we have found that the two types of the postplasmic RNAs are localized in ultra-structurally distinct parts of the postplasm by the electron-microscopic in situ hybridization analysis. These will yield in the systematic knowledge about the RNA localization machanisms in ascidian embryogenesis.