Köller J, Nörskau G, Paul A S, Stuart K, Göringer H U
Laboratorium für Molekulare Biologie, Genzentrum, Martinsried, Germany.
Nucleic Acids Res. 1994 Jun 11;22(11):1988-95. doi: 10.1093/nar/22.11.1988.
RNA editing is a mitochondrial transcript maturation process which evolved in kinetoplastid protozoa. It entails the insertion and deletion of exclusively uridine nucleotides directed by gRNAs into pre-mRNAs. Other participating components are not currently known. The aim of this study was to identify mitochondrial proteins that are in direct physical contact with gRNAs thereby possibly involved in the editing reaction. At low monovalent cation concentration (30 mM KCl) 8 polypeptides with apparent molecular weights ranging from 124 to 9 kDa specifically cross-linked to gRNAs. Three of the proteins, 90, 21, and 9 kDa in size, were able to bind at higher salt concentrations (> or = 100 mM) indicating an enhanced affinity to the gRNA molecules. No cross-links were identified at > or = 250 mM KCl. Four gRNAs, specific for different editing domains of the ATPase 6 and ND7 pre-mRNAs, were in contact with the same set of mitochondrial polypeptides suggesting the assembly of an identical RNP complex that does not include pre-mRNA molecules. The binding of the 90 kDa protein was sensitive to the presence of U-nucleotides at the 3'-end of the gRNAs and could specifically be blocked by modifying free sulfhydryl groups. The interaction with the 124 kDa polypeptide was inhibited by vanadyl ribonucleosides, implicating a role for 2', 3' hydroxyl groups in the gRNA-protein interaction.
RNA编辑是一种在动基体原生动物中进化而来的线粒体转录本成熟过程。它需要由引导RNA（gRNA）将尿苷酸专一性地插入和缺失到前体信使核糖核酸（pre-mRNA）中。目前尚不清楚其他参与成分。本研究的目的是鉴定与gRNA直接发生物理接触从而可能参与编辑反应的线粒体蛋白质。在低单价阳离子浓度（30 mM氯化钾）下，8种表观分子量在124至9 kDa之间的多肽与gRNA特异性交联。其中三种蛋白质，大小分别为90、21和9 kDa，在较高盐浓度（≥100 mM）下能够结合，表明它们对gRNA分子的亲和力增强。在≥250 mM氯化钾时未鉴定到交联。四种分别针对ATP酶6和ND7前体信使核糖核酸不同编辑结构域的gRNA，与同一组线粒体多肽发生接触，这表明形成了一个不包括前体信使核糖核酸分子的相同核糖核蛋白（RNP）复合物。90 kDa蛋白质的结合对gRNA 3'-末端尿苷酸的存在敏感，并且可以通过修饰游离巯基而被特异性阻断。与124 kDa多肽的相互作用受到钒核糖核苷的抑制，这表明2'，3'-羟基在gRNA-蛋白质相互作用中发挥作用。
