Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eIF2C translation initiation factors
Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eIF2C translation initiation factors
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DOI：
10.1016/s0960-9822(02)01394-5
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发表时间：
2003-01-08
期刊：
影响因子：
9.2
通讯作者：
Saigo, K
中科院分区：
文献类型：
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作者：
Doi, N;Zenno, S;Saigo, K
RNA干扰(RNAi)是RNA依赖的长双链转录后基因沉默(PTGS)的过程[1]。在低等真核生物中，被引入细胞质的dsRNA被类似RNaselll的酶DICER切割成21-23个核苷酸的RNA(短干扰[si]RNA)，这可能是靶mRNA降解的指南[2]。在哺乳动物中，依赖长dsRNA的PTGS仅适用于有限数量的细胞类型[3-7]，而在体外合成的siRNA能够有效地诱导多种细胞中的基因沉默[8]。虽然在低等真核生物中的生化和遗传分析表明，DICER和一些PIWI家族成员蛋白是长dsRNA依赖的PTGS所必需的[9-11]，但人们对基于siRNA的PTGS的分子机制知之甚少。在这里，我们证明了属于PIWI家族的DICER和eIF2C翻译起始因子(eIF2C1-4)在哺乳动物siRNA介导的PTGS中发挥着重要作用，很可能是通过协同作用。免疫沉淀实验表明，在人和小鼠细胞中，DICER和eIF2C1或2之间形成了复合体，eIF2C的PIWI结构域是形成这种复合体的关键。
RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS) [1]. In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaselll-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation [2]. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types [3-7], whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells [8]. Although biochemical and genetic analyses in lower eukaryotes; showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS [9-11], little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.