Studies of the interaction of the viral suppressor of RNA silencing protein p19 with small RNAs using fluorescence polarization
Studies of the interaction of the viral suppressor of RNA silencing protein p19 with small RNAs using fluorescence polarization
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DOI：
10.1021/bi800401y
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发表时间：
2008-08-05
期刊：
影响因子：
2.9
通讯作者：
Pezacki, John Paul
中科院分区：
文献类型：
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作者：
Cheng, Jenny;Sagan, Selena M.;Pezacki, John Paul
在感染过程中，tombusvirus使用一种19kda蛋白（p19）作为RNA沉默途径的抑制因子。p19蛋白以二聚体形式与短干扰RNA （siRNA）结合，对短双工RNA具有较高的选择性。由于p19可以结合合成和RNA沉默产生的小RNA，具有较小的序列依赖性和大小选择性，因此该蛋白可作为研究真核生物RNA沉默途径的工具。然而，p19作为研究RNA沉默途径的工具的能力可能会因为其他内源性小RNA（如微RNA (miRNAs)）的存在而变得复杂。为了了解内源性小RNA成分在p19与sirna结合能力方面的重要性，我们研究了p19与人类miR-122的相互作用，miR-122是一种23个核苷酸的双工miRNA，含有几个不匹配的碱基对，在肝脏中含量很高。将其与针对人激酶CSK优化的siRNA的结合特性进行了比较。结合研究是通过荧光极化实验对在RNA链的5'端含有Cy3染料标记的双寡核苷酸以及电泳凝胶迁移率转移实验进行的。两种方法都表明，合成的碱基配对无错配的siRNA比miR-122的选择性高3倍。我们的研究结果表明，p19可以区分sirna和miRNA物种，尽管结合常数的差异并不大，与内源性miRNA的相互作用可以完全忽略。
Tombusviruses use a 19 kDa protein (p19) as a suppressor of the RNA silencing pathway during infection. The p19 protein binds to short-interfering RNA (siRNA) as a dimer and shows a high selectivity for short duplex RNAs over other RNA species. Since p19 can bind to synthetic and RNA silencing generated small RNAs with little sequence dependence and with size selectivity, this protein has utility as a tool for studying RNA silencing pathways in eukaryotes. However, the ability of p19 to serve as a tool for studying RNA silencing pathways may be complicated by the presence of other endogenous small RNAs such as micro-RNAs (miRNAs). To understand the importance of endogenous small RNA components with respect to p19's ability to bind to siRNAs, we examined the interactions of p19 with human miR-122, a 23-nucleotide duplex miRNA containing several mismatched base pairs that is highly abundant in the liver. The binding characteristics were compared with those of an siRNA optimized against the human kinase CSK. The binding studies were performed using fluorescence polarization experiments on duplex oligonucleotides containing Cy3 dye labels at the 5'-end of one of the strands of RNA as well as electrophoretic gel mobility shift assays. Both methods indicate that the synthetic siRNA with no mismatches in base pairing bound with > 3-fold selectivity over that of miR-122. Our results suggest that p19 can distinguish between siRNAs and miRNA species, although the difference in binding constants is not so large that interactions with endogenous miRNAs can be totally ignored.