RNA transport to the vegetal cortex of Xenopus oocytes.
RNA transport to the vegetal cortex of Xenopus oocytes.
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DOI：
10.1006/dbio.1996.0249
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发表时间：
1996-10
影响因子：
2.7
通讯作者：
Yi Zhou;M. L. King
中科院分区：
文献类型：
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作者：
Yi Zhou;M. L. King
Xcat-2 RNA 是非洲爪蟾种质的一个组成部分，与线粒体云物质一起定位到 II 期卵母细胞的植物皮层。 Vg1 RNA 也定位于植物皮层，但随后使用微管依赖性途径定位于 III/IV 期卵母细胞。为了进一步分析 RNA 转运所涉及的机制，使用原位杂交和放射自显影技术跟踪 IV 期卵母细胞中内源 Vg1 和注射的 Xcat-2 转录本的定位。我们证明 Xcat-2 能够完全独立于线粒体云定位到植物皮层。 Xcat-2 RNA 似乎使用晚期 Vg1 定位途径，因为冷或诺考达唑处理使微管解聚，阻止了 Xcat-2 转录物的易位，但不会导致锚定在皮质中的 Xcat-2 的破坏。此外，Vg1 和 Xcat-2 RNA 的 RNA 运输均表现出阶段依赖性，因为它们在注射后并不定位于完全生长的 VI 阶段卵母细胞中。将 Xcat-2 引导至植物皮层所需且足够的 RNA 序列被映射到紧邻开放阅读框的 150 nt 序列以及 3' 非翻译区末端的附加序列。通过将缺失突变体转录物注射到 IV 期卵母细胞中并通过 RNase 保护和放射自显影监测定位来完成绘图。所有能够进行易位的突变体也能够进行皮质锚定，这表明这两个步骤使用了相同的信号。我们推测爪蟾卵子发生过程中进化出了两条独立的 RNA 途径。一种专门用于通过线粒体云运输种质的途径很早就发生，以确保生殖细胞谱系的分离。另一条晚期途径可能作为更通用的转运系统，用于定位参与体细胞分化的 RNA。
Xcat-2 RNA, a component of the germ plasm in Xenopus, localizes with the mitochondrial cloud material to the vegetal cortex in stage II oocytes. Vg1 RNA also localizes to the vegetal cortex, but later in stage III/IV oocytes, using a microtubule dependent pathway. To further analyze the mechanisms involved in RNA transport, in situ hybridization and autoradiography were used to follow the localization of endogenous Vg1 and injected Xcat-2 transcripts in stage IV oocytes. We show that Xcat-2 is competent to localize to the vegetal cortex quite independently of the mitochondrial cloud. Xcat-2 RNA appears to use the late Vg1 localization pathway, as depolymerization of microtubules by cold or nocodazole treatment prevented translocation of Xcat-2 transcripts, but did not result in the disruption of Xcat-2 anchored in the cortex. Furthermore, RNA transport was shown to be stage dependent for both Vg1 and Xcat-2 RNAs, as they did not localize in fully grown stage VI oocytes after injection. RNA sequences both required and sufficient to direct Xcat-2 to the vegetal cortex were mapped to a sequence of 150 nt immediately adjacent to the open reading frame and additional sequences at the end of the 3' untranslated region. Mapping was accomplished by injecting deletion mutant transcripts into stage IV oocytes and monitoring localization by RNase protection and autoradiography. All mutants competent for translocation were also capable of cortical anchoring, suggesting that the same signal is used for both steps. We speculate that two separate RNA pathways evolved during the course of Xenopus oogenesis. One pathway, specialized for the transport of germ plasm by way of the mitochondrial cloud, occurs early to ensure the segregation of the germ cell lineage. The other, late, pathway may serve as the more general transport system for localizing RNAs involved in somatic cell differentiation.