Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.
Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.
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DOI：
10.1083/jcb.103.3.699
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发表时间：
1986-09
期刊：
影响因子：
--
通讯作者：
Rome LH
中科院分区：
文献类型：
--
作者：
Kedersha NL;Rome LH
大鼠肝包被囊泡制剂中经常发现含有小的卵圆形小体，在形态上与包被囊泡相似。我们用蔗糖密度梯度和制备性琼脂糖凝胶电泳法将这些小体纯化为均一。当负染和电子显微镜观察时，纯化的结构显示出非常明显和复杂的形态，类似于形成大教堂拱顶的多个拱门。它们的尺寸为35×65纳米，因此比核糖体大得多。当受到SDS PAGE分析时，这些结构，我们称为拱顶，似乎包含几个小的和五个主要的物种：Mr 210,000,192,000,104,000，54,000和37,000。其中一种(mr为104,000)在很大程度上占优势，占考马斯亮蓝蛋白总量的70%以上。MR 37,000的另一个主要物种已被鉴定为具有不寻常碱基组成的小RNA物种(腺苷12.0%，鸟苷29.7%，尿苷30.9%，胞苷27.4%)，在尿素PAGE中作为单一物种在5S和5.8S核糖体标准之间迁移，包含约140个碱基。虽然RNA只占整个结构的4.6%，但由于颗粒的大小，每个颗粒需要包含大约9个这种RNA分子。针对整个颗粒制备的抗体主要针对主要的(mR为104,000)多肽种类。虽然它们不直接与Western blotts上的RNA成分反应，但这些抗体从代谢标记的大鼠肝癌细胞中免疫沉淀出大小相同的32P标记的RNA。从人成纤维细胞、小鼠3T3细胞、神经胶质细胞和兔肺泡巨噬细胞的部分纯化组分中观察到拱顶。因此，这些新的核糖核蛋白结构似乎广泛分布在不同的细胞类型中。金库的功能目前尚不清楚。
Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue- staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown.