Shi H, O'Brien C A, Van Horn D J, Wolin S L
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
RNA. 1996 Aug;2(8):769-84.
In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.
在脊椎动物和无脊椎动物细胞中，60 kDa的Ro自身抗原都与被称为Y RNA的小细胞质RNA结合。在非洲爪蟾卵母细胞中，60 kDa的Ro蛋白还与一类含有内部突变的5S rRNA前体形成复合物。由于这些5S rRNA前体的加工效率低下并最终被降解，Ro蛋白可能在5S rRNA生物合成的质量控制途径中发挥作用。我们研究了赋予60 kDa Ro蛋白结合能力的突变型5S rRNA中的序列和二级结构决定因素。突变型5S rRNA折叠形成一种替代螺旋，这是60 kDa Ro蛋白识别所必需的。破坏替代螺旋的突变会消除Ro蛋白的结合，而恢复螺旋的补偿性变化则能被Ro蛋白有效结合。当使用硫酸二甲酯和寡核苷酸定向RNase H切割来探测突变RNA的结构时，结果与替代结构的形成一致。La蛋白也与突变型5S rRNA前体形成复合物，它与60 kDa Ro蛋白一样能保护相似序列免受核酸酶消化。因此，这两种蛋白的结合位点要么在RNA上相邻，要么这两种蛋白可能通过蛋白质-蛋白质相互作用形成复合物。当人Ro蛋白在酿酒酵母中表达时，该蛋白能结合野生型5S rRNA前体，这表明野生型前体群体也会折叠成替代结构。
