Sioud M
Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway.
J Mol Biol. 1994 Oct 7;242(5):619-29. doi: 10.1006/jmbi.1994.1612.
Ribozymes are RNA molecules that cleave other RNA molecules. Thus, ribozymes offer a new way of inhibiting expression of specific genes whose nucleotide sequences are known. Intracellular stability of ribozymes is an important factor for their efficacy. We previously showed that hammerhead ribozyme directed against mRNA of tumour necrosis factor alpha (TNF alpha) slowly acquires resistance to degradation in cultured human cells. In order to explain this resistance, we now report on endogenous cellular protein(s) that bind to TNF alpha-ribozyme (TNF alpha-Rz) in solution to form stable complexes during native gel electrophoresis. Suppression of the effects of ribonucleases in the cytoplasmic extracts allowed approximately 80% of the input ribozyme RNA to be recovered in the form of complexes, indicating that complex formation protected the ribozyme from degradation. Treatment of the ribozyme-protein complexes with proteinase K prior to electrophoresis led to the recovery of full-length ribozyme. Interestingly, ribozyme-protein complexes retained cleavage activity, suggesting that the binding is in reversible equilibrium. Analysis of protein cytoplasmic extracts for binding to sub-fragments of TNF alpha-Rz demonstrated that protein binds to a conformational epitope formed by an interaction between the 5' end of TNF alpha-Rz and its catalytic domain. Competition of the ribozyme-protein binding with a ribozyme construct containing DNA instead of RNA at the 5' end, indicated that the ribose phosphate backbone of the 5' end is required for strong binding. The protein responsible for the formation of the complex with low electrophoretic mobility was found to be specific for the TNF alpha-Rz, since ribozyme for HIV-1 integrase gene (Int-Rz), or for human interleukin-2 (IL2-Rz) did not compete significantly with the TNF alpha-Rz binding. Covalent linkage of the IL2-Rz to the 3' end of TNF alpha-Rz, or to the proposed RNA protein binding site conferred protein binding and enhanced the stability and activity of the chimeric molecules. The RNA epitope identified in this study, through its endogenous protein binding, may serve as an oligonucleotide cassette for enhancing the in vivo stability and activity of other RNA molecules in general. This RNA epitope will also be useful in the study of RNA-protein interactions.
核酶是能切割其他RNA分子的RNA分子。因此，核酶为抑制已知核苷酸序列的特定基因的表达提供了一种新方法。核酶的细胞内稳定性是其发挥功效的一个重要因素。我们之前表明，针对肿瘤坏死因子α（TNFα）mRNA的锤头状核酶在培养的人类细胞中对降解的抗性逐渐增强。为了解释这种抗性，我们现在报告一种内源性细胞蛋白，它在天然凝胶电泳过程中与溶液中的TNFα核酶（TNFα-Rz）结合形成稳定复合物。抑制细胞质提取物中核糖核酸酶的作用使得大约80%的输入核酶RNA以复合物的形式被回收，这表明复合物的形成保护了核酶不被降解。在电泳前用蛋白酶K处理核酶-蛋白质复合物导致全长核酶的回收。有趣的是，核酶-蛋白质复合物保留了切割活性，这表明这种结合处于可逆平衡。对蛋白质细胞质提取物与TNFα-Rz亚片段结合的分析表明，蛋白质与由TNFα-Rz 5'端与其催化结构域之间相互作用形成的构象表位结合。在5'端用含DNA而非RNA的核酶构建体竞争核酶-蛋白质结合，表明5'端的核糖磷酸骨架对于强结合是必需的。发现与低电泳迁移率复合物形成相关的蛋白质对TNFα-Rz具有特异性，因为针对HIV-1整合酶基因的核酶（Int-Rz）或针对人白细胞介素-2的核酶（IL2-Rz）与TNFα-Rz结合没有明显竞争。将IL2-Rz共价连接到TNFα-Rz的3'端或拟议的RNA-蛋白质结合位点赋予了蛋白质结合能力，并增强了嵌合分子的稳定性和活性。通过其与内源性蛋白质结合在本研究中鉴定出的RNA表位，一般来说可能作为一种寡核苷酸盒来增强其他RNA分子在体内的稳定性和活性。这种RNA表位在RNA-蛋白质相互作用的研究中也将是有用的。
