Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells
Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells
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DOI：
10.1074/jbc.m010570200
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发表时间：
2001-05-04
影响因子：
4.8
通讯作者：
Taira, K
中科院分区：
文献类型：
--
作者：
Kato, Y;Kuwabara, T;Taira, K
为了研究核酶在体外和体内的作用，合成了19个不同的功能RNA，在两种不同的启动子tRNA或U6的控制下，这些RNA在培养细胞中的活性与它们各自的体外化学切割反应的动力学参数之间没有发现相关性，表明在任何情况下，化学切割都不是体内的限速步骤，例如丁型肝炎病毒(HDV)核酶，其体外活性几乎比锤头状核酶低3个数量级，但在使用适当的表达系统时，在细胞中仍表现出类似的活性。正如预期的那样，其作用依赖于核RNaseP的外部引导序列在细胞核中更活跃。对培养细胞的数据分析清楚地表明，细胞质中的核酶明显比核中的核酶活性高，这表明细胞质中的成熟mRNAs可能比细胞核中的前mRNAs更容易与反义分子结合。我们的发现将对未来细胞内活性功能分子的设计有所帮助。
Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus, No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo, For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.