Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens
Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens
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DOI：
10.1073/pnas.1402353111
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发表时间：
2014-03-25
影响因子：
11.1
通讯作者：
von Mering, Christian
中科院分区：
文献类型：
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作者：
Franceschini, Andrea;Meier, Roger;von Mering, Christian
在人类细胞中进行系统的遗传扰动筛查在技术上仍然具有挑战性。通常，使用大量化学合成的siRNA寡核苷酸文库，每个文库都旨在通过RNA干扰(RNAi)机制降解特定的细胞mRNA。在这里，我们报告了来自三个全基因组siRNA筛查的数据，这些筛查是为了揭示两种细菌和一种病毒病原体感染人类细胞所需的宿主因素。我们发现，siRNAs的大多数表型效应与预期的“靶向”机制无关，该机制是由21-nT siRNA序列与靶mRNA的完全互补定义的。相反，表型在很大程度上是由siRNAs通过“种子”区域(即核苷酸2-8)与多个mRNAs部分互补而产生的“非靶标”效应，这让人想起了内源性microRNAs的特异性被确定的方式。定量分析使得能够预测出强烈和特定地阻止感染的种子，而与预期的靶标效应无关。通过设计完全没有任何靶上序列匹配的寡核苷酸，但可以强烈地复制预测的表型，这一预测得到了实验证实。我们的结果表明，已发表的RNAi筛选主要无意地筛选了microRNA种子的序列空间，而不是预期的蛋白质编码基因的靶标空间。这有助于解释为什么之前发表的RNAi屏幕显示的重叠相对较少。我们的分析建议了一种可能的方法来识别“种子试剂”来控制感兴趣的表型，并建立了从过去和未来的RNAi筛查中提取有价值的未利用信息的一般策略。
Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.