Matsumoto K, Aoki K, Dohmae N, Takio K, Tsujimoto M
RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan.
Nucleic Acids Res. 2000 Dec 1;28(23):4689-97. doi: 10.1093/nar/28.23.4689.
In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)-cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.
为了分离mRNA结合蛋白，我们通过寡聚（dT）-纤维素色谱法对非洲爪蟾卵母细胞裂解液进行了分级分离。一种20 kDa的蛋白质是洗脱液的主要成分。cDNA克隆显示，这种蛋白质是冷诱导RNA结合蛋白（CIRP）的非洲爪蟾同源物，CIRP最初在哺乳动物细胞中被鉴定为一种在温度下降时过度表达的蛋白质。这种非洲爪蟾蛋白质，在这里称为xCIRP2，在成年非洲爪蟾组织的卵巢、睾丸和大脑中高度表达。在卵母细胞中，它主要定位于细胞质中。通过生化分级分离，我们提供证据表明xCIRP2与核糖体相关，这表明它参与卵母细胞中的翻译调控。将标记的mRNA显微注射到卵母细胞中，然后对卵母细胞裂解液进行紫外线交联，从而鉴定出两种主要的RNA结合活性。对RNA结合蛋白进行免疫沉淀表明，一种是xCIRP2，另一种含有FRGY2。FRGY2是参与母源mRNA翻译掩盖的mRNA储存颗粒的主要成分之一，其RNA结合结构域与细菌冷休克蛋白的结构域保守。本文讨论了真核和原核类型的冷休克RNA结合蛋白在卵母细胞中高丰度表达的可能意义。
