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

早期海鞘胚胎质后RNA定位机制分析。

• 批准号: 12680714
• 负责人: MAKABE Kazuhiro
• 金额: $ 2.37万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680714/
• 关键词: ascidian; mnternal factors; RNA; lacalization; postplasm; UTR; EST; 母性RNA; 局在RNA; 細胞内局在

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.

我们在海鞘卵中分离到了许多RNA，其中一些RNA位于海鞘卵的后部区域，被认为是胚胎发育过程中发育轴的特化和不均等卵裂模式所必需的，因此被命名为后质RNA。本研究的目的是通过鉴定控制其定位的顺式元件和与元件结合的反式作用因子来阐明asicdian卵和胚胎中RNA分选的分子机制。通过分析上述6种RNA的详细RNA分布模式，我们发现它们根据定位途径分为两组; I型和II型。注射标记的后质RNA的合成RNA显示，它们的31个UTR序列对于RNA在胚胎中的正确定位是必需的和足够的，因为这两种类型的RNA。此外，我们已经将区域最小化到约50 nt。为了鉴定和表征与该区域结合的反式作用因子，我们正在进行凝胶移位实验、East-Western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鸡蛋中序列特异性RNA结合蛋白。同时，RNA结合蛋白的生物化学纯化和编码蛋白质的cDNA的分离也在进行中。另一方面，我们已经发现，这两种类型的postplasma RNA定位在超微结构不同的部分postplasma的电子显微镜原位杂交分析。这将有助于对海鞘胚胎发生中的RNA定位机制有系统的认识。

项目成果

T.Kawashima: "Update of MAGEST : MAboya Gene Expression Patterns and Sequence Tags"Nuc. Acids Res.. 30. 119-120 (2002)

T.Kawashima：“MAGEST 更新：MAboya 基因表达模式和序列标签”Nuc。

K. Yagi.: "Retinoic acid differently affects the formation of palps and surrounding neurons in the ascidian tadpole"Dev. Genes Evol.. (in press). (2002)

K. Yagi.：“视黄酸对海鞘蝌蚪触须和周围神经元的形成有不同的影响”Dev.

K.W.Makabe: "Matend genetic information stored in fertilized eggs of the ascidinn, Halocyathin roretzi"Biology of ascidian. (in press). (2001)

K.W.Makabe：“Matend 遗传信息存储在海鞘的受精卵中，Halocyathin roretzi”海鞘生物学。

Y. Sasakura.: "Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos"Dev Biol.. 220. 365-378 (2000)

Y. Sasakura.：“早期海鞘胚胎后植物细胞质中母体 RNA 定位的两种途径”Dev Biol.. 220. 365-378 (2000)

K. W. Makabe.: "Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for gene expression catalog of ascidian development"Development. 128. 2555-2567 (2001)

K. W. Makabe.：“Halocynthia roretzi 卵中母体遗传信息的大规模 cDNA 分析，用于海鞘发育的基因表达目录”开发。