Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum
Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum
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DOI：
10.1091/mbc.e04-03-0265
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发表时间：
2004-10-01
影响因子：
3.3
通讯作者：
King, ML
中科院分区：
文献类型：
--
作者：
Chang, P;Torres, J;King, ML
非洲爪蟾的生殖细胞谱系由起源于卵母细胞中独特的线粒体云（MC）内的种质遗传所指定。种质含有涉及生殖细胞发育的局部RNA，包括Xcat 2和Xdazl。为了了解RNA定位于MC的早期途径的机制，我们将实时共聚焦成像和光漂白分析应用于显微注射荧光Xcat 2和Xdazl RNA构建体的卵母细胞。这些RNA通过扩散均匀分散在整个细胞质中，然后逐渐固定并在MC中形成聚集体。截留在MC中没有阻止微管破坏，并不需要本地化的germinium颗粒。如Dil（16）（3）标记和免疫荧光分析所示，固定化RNA构建体与MC中密集的内质网（ER）共分布并显示协调运动。Vg 1 RBP/Vera蛋白参与了晚期通路RNA与植物ER的连接，显示其特异性结合野生型Xcat 2 3'非翻译区和定位缺陷构建体。我们发现内源性Vg 1 RBP/维拉和Vg 1 RBP/维拉绿色荧光蛋白在很大程度上被排除在MC，但随后与Xcat 2和ER在植物皮质共分布。我们得出结论，生殖系RNA本地化到MC通过扩散/截留机制，涉及Vg 1 RBP/维拉独立协会与ER。
The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil(16)(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.