Analysis of RNA sorting mechanisms in the ascidian embryos.

我们已经分离到许多定位在海鞘虫卵中的RNA。值得注意的是，在海鞘卵子中发现了作为局部功能分子的RNA，这些卵子被称为“马赛克”卵子。我们研究的六个RNA定位于卵子的后部植物区，称为后质。据报道，该区域参与了早期卵裂球前后轴的形成和不等卵裂的产生。因此，我们将这些RNA命名为质后RNA。对它们的表达模式的详细分析表明，根据它们的运输途径，它们被分为两类(类型I和类型II)。将标记的合成RNA显微注射到未受精卵中的实验表明，RNAs的3‘UTR区中的小分子对于正确的定位是必要的和充分的。在这些区域没有明显的保守序列，但注射实验显示。使用3‘UTRs的一部分确定了负责定位的50个核苷酸元件。为了研究海囊虫卵和胚胎中RNA分选的分子机制，我们目前正在尝试利用凝胶移动、东西和紫外光交联技术从海鞘虫卵提取液中分离与已鉴定元件结合的序列特异性RNA结合蛋白，并对这些蛋白的生化特性进行分析。另一方面，我们在电子显微镜水平上揭示了后质质RNA分布在胚胎CAB区的特定位置。这些结果将有助于揭示海鞘虫卵和胚胎中RNA分选的整个分子机制。

We have isolated many RNAs localized in the ascidian eggs. It is noteworthy to have found RNAs as localized functional molecules in the ascidian eggs, which are known to be "mosaic" eggs. Six RNAs we studied are localized at the posterior-vegetal region of the egg, named the postplasm. This region is reported to be involved in the anterior-posterior axis formation and generation of unequal cleavages of early blastomeres. We, therefore, designated these RNAs the postplasmic RNAs. The detailed analysis of their expression patterns revealed that they are categorized into two groups (type I and type II) based on their transport pathways. Microinjection experiments of labeled synthetic RNAs into the unfertilized eggs suggested that small elements in the 3 'UTR of the RNAs are necessary and sufficient to proper localization. There is no obvious conserved sequence in the regions, but the injection assay. using portions of the 3'UTRs identified 50-nucleotide elements responsible for the localization. In order to investigate molecular mechanisms of RNA sorting in the ascidian eggs and embryos, we are currently trying to isolate sequence-specific RNA-binding proteins which bind to the identified elements from the ascidian egg extract using gel shift, east-western and UV-cross link techniques and analyze on the biochemical characteristics of the proteins. On the other hand, we revealed that the postplasmic RNAs distribute at specific sites in the CAB region of the embryo at the electron microscopic level. These will shed a light to the whole molecular mechanisms on the RNA sorting in the ascidian eggs and embryos.