Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis
Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis
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DOI：
10.1186/1471-2164-11-175
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发表时间：
2010-03-15
期刊：
影响因子：
4.4
通讯作者：
Dunham, Ian
中科院分区：
文献类型：
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作者：
Sudbery, Ian;Enright, Anton J.;Dunham, Ian
背景：siRNA 抑制 RNA 是鉴定特定生物过程所需基因的常用方法。然而，使用 siRNA 进行的 RNAi 筛选受到 siRNA 的非特异性或脱靶效应的阻碍，使得很难将真正的命中与假阳性区分开。据认为，RNAi 实验中出现的许多脱靶效应是由于 siRNA 作为 microRNA (miRNA) 发挥作用，通过与 6 或 7 nt“种子”序列匹配，导致非预期靶标的基因表达减少。我们在 TRAIL 细胞凋亡诱导途径的新型调节剂的 siRNA 筛选过程中对脱靶效应进行了仔细检查。结果：我们鉴定了 3 个六聚体和 3 个七聚体种子序列，它们在 TRAIL 细胞凋亡筛选中的前 20 个 siRNA 中多次出现。使用一种新颖的统计富集方法，我们系统地鉴定了另外 17 个六聚体和 13 个七聚体种子序列，这些序列富含高分 siRNA。这些种子序列之一的存在（可以解释 8 个已确认的脱靶效应中的 6 个）足以引发表型。其中三个种子序列出现在人类 miRNA miR-26a、miR-145 和 miR-384 中。转染这些 miRNA 的模拟物可保护多种细胞类型免遭 TRAIL 诱导的细胞死亡。结论：我们已经证明了 miR-26a、miR-145 和 miR-26a 在 TRAIL 诱导的细胞凋亡中的作用。此外，这些结果表明 RNAi 筛选丰富了具有相关脱靶效应的 siRNA。其中一些效应可以通过高分 siRNA 中某些种子序列的过度表达来识别，我们证明了这种对富集种子序列进行系统分析的有用性。
Background: RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s).Results: We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death.Conclusions: We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.