Rashid Rumana, Aittaleb Mohamed, Chen Qiong, Spiegel Katharina, Demeler Borries, Li Hong
Department of Chemistry and Biochemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
J Mol Biol. 2003 Oct 17;333(2):295-306. doi: 10.1016/j.jmb.2003.08.012.
Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.
C/D 盒小核糖核蛋白颗粒（sRNP）是真核生物中核仁小核糖核蛋白颗粒（snoRNP）的古菌同源物，负责核糖体RNA和转运RNA的位点特异性2'-O-甲基化。C/D 盒 sRNP 的功能特征是三种核心蛋白围绕 C/D 盒 RNA 逐步组装，这些核心蛋白包括纤维蛋白原、Nop5p 和 L7Ae。所有 C/D 盒 RNA 中最显著的结构特征是存在两个保守的 C/D 盒基序，通常还伴有一个，有时是两个反义元件，位于 D 盒或 D' 盒上游紧邻位置。尽管反义元件分布不对称，但 C/D 盒基序的二分特征似乎与最近报道的纤维蛋白原和 Nop5p 之间核心蛋白复合物的三维结构非常吻合。本文研究了 C/D 盒 RNA 和纤维蛋白原 - Nop5p 复合物中对称特征的功能意义。采用定点诱变来生成缺失两个 C/D 盒基序之一的 C/D 盒 RNA，以及生成自我缔合缺陷的突变纤维蛋白原 - Nop5p 复合物。通过凝胶迁移率变动分析、分析型超速离心和体外催化研究评估突变组分的组装能力和指导甲基转移反应的能力。本文给出的结果表明，虽然 C/D 盒 sRNP 能够不对称组装，但 C/D 盒 RNA 和纤维蛋白原 - Nop5p 复合物中的对称性对于高效催化都是必需的。这些发现强调了功能组装在甲基转移反应中的重要性。
