Analysis of RNA sorting mechanisms in the ascidian embryos.

我们已经分离出许多定位于海鞘卵的rna。值得注意的是，在被称为“马赛克”卵的海鞘卵中发现了rna作为局部功能分子。我们研究的六种rna定位于卵的后植物区，称为后质。据报道，该区域参与早期卵裂球前后轴的形成和不均匀劈裂的产生。因此，我们将这些rna命名为质后rna。对其表达模式的详细分析表明，根据其运输途径，它们可分为两类（I型和II型）。将标记的合成rna注入未受精卵的显微注射实验表明，rna的3 'UTR中的小元素对于正确定位是必要和充分的。在区域内没有明显的保守序列，但注射测定。利用部分3' utr鉴定出负责定位的50个核苷酸元素。为了研究海鞘卵和胚胎中RNA分选的分子机制，我们目前正尝试利用凝胶转移、东西转移和紫外交联技术从海鞘卵提取液中分离序列特异性RNA结合蛋白，并对其生化特性进行分析。另一方面，我们在电镜水平上揭示了质后rna分布在胚胎CAB区域的特定位点上。这将有助于揭示海鞘卵和胚胎中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.