An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division
An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division
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DOI：
10.1038/nature03159
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发表时间：
2004-12-23
期刊：
影响因子：
64.8
通讯作者：
Buchholz, F
中科院分区：
文献类型：
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作者：
Kittler, R;Putz, G;Buchholz, F
RNA干扰（RNAi）是一种进化上保守的防御机制，通过降解信使RNA特异性沉默基因;该过程由同源双链（ds）RNA分子介导（1-4）。在无脊椎动物中，长dsRNA已用于全基因组筛选，并提供了对基因功能的见解（5-8）。由于长双链RNA在许多脊椎动物中触发非特异性干扰素反应，因此必须对这些生物体使用短干扰（si）RNA或短发夹（sh）RNA以确保特异性基因沉默（9-11）。在这里，我们报告了从代表15，497个人类基因的序列验证的互补DNA集合中产生内切核糖核酸酶制备的短干扰（esi）RNA 12的基因组规模文库。我们使用来自该文库的5，305个esiRNA来筛选HeLa细胞中细胞分裂所需的基因。使用一个主要的高通量细胞活力筛选，其次是二级高内容视频显微镜分析，我们确定了37个细胞分裂所需的基因。这些包括几个剪接因子，其敲低产生有丝分裂纺锤体缺陷。此外，一个假定的核输出终止子被发现，以加快细胞增殖和有丝分裂的进程后，敲低。因此，我们的研究揭示了细胞分裂的新方面，并建立了esiRNA作为哺乳动物细胞中基因组RNAi筛选的通用方法。
RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds) RNA molecules(1-4). In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions(5-8). Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering ( si) RNA or short hairpin (sh) RNAs must be used for these organisms to ensure specific gene silencing(9-11). Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi) RNAs12 from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.