Piñol-Roma S
Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Mol Biol Cell. 1999 Jan;10(1):77-90. doi: 10.1091/mbc.10.1.77.
rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin-a major nucleolar RNA-binding protein-contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.
随着前体rRNA从转录机制中产生，rRNA前体在其整个长度上都与特定蛋白质结合。前体rRNA与蛋白质作为核糖核蛋白（RNP）复合物的结合在18S、5.8S和28S rRNA成熟过程中持续存在，并贯穿于核仁中核糖体亚基的组装过程。核糖体前体RNP复合物除了包含核糖体蛋白外，还含有数量未知的非核糖体核仁蛋白，以及小核仁RNA-核糖核蛋白（sno-RNP）。本报告描述了使用一种特定、快速且温和的免疫纯化方法来分离和分析包含非核糖体核仁蛋白、核糖体蛋白和rRNA的人类RNP复合物。用针对核仁素（一种主要的核仁RNA结合蛋白）的抗体免疫纯化的复合物包含几种不同的特定多肽，除核仁素外，还包括先前鉴定的核仁蛋白B23和纤维原蛋白、具有核糖体蛋白电泳迁移特征的蛋白质（包括核糖体蛋白S6），以及许多其他未鉴定的蛋白质。这些蛋白质之间的物理结合主要由RNA介导，因为复合物在用核糖核酸酶消化后会解离。从M期细胞中分离的复合物在蛋白质组成上与从间期细胞核提取物中分离的复合物相似。因此，尽管在M期rRNA合成和加工停止，但在间期与核仁素结合的主要蛋白质在有丝分裂期间仍保留在RNP复合物中。此外，前体rRNA以及加工后的18S和28S rRNA以及候选rRNA加工中间体，都与免疫纯化的复合物相关联。因此，这里描述的rRNP复合物的特征表明它们代表成熟细胞质核糖体亚基的真正前体。
