Comparison of siRNA-induced off-target RNA and protein effects
Comparison of siRNA-induced off-target RNA and protein effects
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DOI：
10.1261/rna.352507
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发表时间：
2007-03-01
期刊：
影响因子：
4.5
通讯作者：
Sharp, Phillip A.
中科院分区：
文献类型：
--
作者：
Aleman, Lourdes M.;Doench, John;Sharp, Phillip A.
通过对转染短干扰RNA（siRNA）后的微阵列结果进行生物信息学分析，已经观察到许多mRNA的下调。这些mRNA变化中的许多是由于siRNA引导链与部分互补位点的相互作用，因此被认为是“脱靶”效应。为了检查mRNA：siRNA相互作用对脱靶效应的重要性，我们产生了一组mRNA：siRNA组合，其在位于整合的报告基因的3' UTR中的两个siRNA结合位点的第9、第10和第11位中含有单错配和双错配、凸起和非典型碱基配对相互作用。大约一半的在位置9-11中含有错配的mRNA：siRNA组合导致mRNA减少两倍或更多，伴随不同程度的蛋白质敲低。然而，各种mRNA：siRNA组合的mRNA和蛋白质分析揭示了mRNA和蛋白质水平不相关的情况。从不完全互补的siRNA与内源基因的相互作用中回收的所得降解产物的分析揭示了一小部分产物映射到典型的siRNA切割位点。此外，ARGONAUTE 2（AGO 2）的下调，唯一的AGO家族蛋白已知催化典型的siRNA介导的切割，并没有显着影响mRNA敲低的程度观察到的一个稳定表达的报告转染后，不完全互补的siRNA。这些结果表明，尽管在siRNA和脱靶转录物之间可以发生一定程度的典型siRNA切割，但大多数脱靶mRNA的减少可能归因于AGO 2非依赖性降解过程。
The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered "off-target'' effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3' UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA: siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resulting degradation products recovered from an imperfectly complementary siRNA interaction with an endogenous gene reveals a small fraction of products that map to the canonical siRNA cleavage site. Furthermore, downregulation of ARGONAUTE 2 (AGO2), the only AGO family protein known to catalyze canonical siRNA-mediated cleavage, did not significantly affect the degree of mRNA knockdown observed for one of the stably expressed reporters after transfection of an imperfectly complementary siRNA. These results indicate that although some degree of canonical siRNA cleavage can take place between a siRNA and an off-target transcript, most off-target mRNA reductions are likely attributable to AGO2-independent degradation processes.