Schwartz S P, Aisenthal L, Elisha Z, Oberman F, Yisraeli J K
Department of Anatomy and Embryology, Hebrew University Medical School, Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11895-9. doi: 10.1073/pnas.89.24.11895.
Vg1 mRNA, a maternal message encoding a member of the transforming growth factor beta superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nucleotide sequence has been identified in Vg1 RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, 991-994]. To understand how cis- and trans-acting factors are involved in Vg1 mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vg1 mRNA. S100 extracts of late-stage oocytes contain a protein-binding activity that protects specific regions of labeled Vg1 mRNA from degradation by RNase T1. The use of different regions of Vg1 RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vg1 message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the down-stream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor beta 5 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.
Vg1信使核糖核酸（mRNA）是一种母源信使，编码转化生长因子β超家族的一个成员，在非洲爪蟾卵母细胞发生的一个狭窄时期内定位于植物皮层。在Vg1核糖核酸中已鉴定出一段340个核苷酸的序列，该序列指导其植物定位[莫里，K.L. & 梅尔顿，D.A.（1992年）《科学》255卷，991 - 994页]。为了解顺式和反式作用因子如何参与Vg1信使核糖核酸的定位，我们在体外寻找卵母细胞蛋白与Vg1信使核糖核酸之间的特异性相互作用。晚期卵母细胞的S100提取物含有一种蛋白结合活性，可保护标记的Vg1信使核糖核酸的特定区域不被核糖核酸酶T1降解。在竞争反应中使用Vg1核糖核酸的不同区域揭示了两个结合位点，均位于Vg1信使核糖核酸3'非翻译区的前半部分。紫外线交联主要标记一种69千道尔顿的蛋白；饱和分析和竞争研究表明，该蛋白与下游位点高亲和力结合，该位点对应于340个核苷酸的植物定位序列。与该区域的结合受到另一种植物定位的信使核糖核酸——转化生长因子β5的抑制，但不受动物定位的核糖核酸An2的抑制。这些数据表明，植物定位的信使核糖核酸共享一个结合基序，通过特定的核糖核酸 - 蛋白相互作用帮助它们实现细胞内分布。
