Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages
Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages
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DOI：
10.1093/nar/gkv143
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发表时间：
2015-03-11
影响因子：
14.9
通讯作者：
Crooke, Stanley T.
中科院分区：
文献类型：
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作者：
Liang, Xue-hai;Sun, Hong;Crooke, Stanley T.
尽管化学修饰的反义寡核苷酸(ASOS)抑制基因表达的RNaseH依赖机制已经被很好地描述，但关于ASOS与细胞内蛋白之间的相互作用可能改变ASOS的细胞定位和/或效力的了解很少。在这里，我们报告了使用多步亲和选择方法鉴定56个细胞内ASO结合蛋白。许多被测试的蛋白质对ASO活性没有显著影响；然而，包括La/SSB、NPM1、ANXA2、VARS和PC4在内的一些蛋白质似乎通过与亚细胞分布有关的机制来增强ASO活性。VARS和ANXA2与ASO共定位于胞内细胞器，VARS水平的降低改变了溶酶体/ASO的定位模式，提示这些蛋白可能促进ASO从内吞途径释放。La和NPM1的耗竭降低了核ASO水平，暗示了ASO核积累的潜在作用。另一方面，Ku70和Ku80蛋白抑制ASO活性，很可能是通过与RNaseH1竞争ASO/RNA双链结合而实现的。我们的结果表明，硫代修饰的ASO结合了一系列细胞蛋白，这些蛋白通过不同的机制影响ASO活性。
Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms.