Argonaute Pull-Down and RISC Analysis Using 2'-0-Methylated Oligonucleotides Affinity Matrices
Argonaute Pull-Down and RISC Analysis Using 2'-0-Methylated Oligonucleotides Affinity Matrices
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DOI：
10.1007/978-1-61779-046-1_16
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Simard, Martin J.
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文献类型：
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作者：
Jannot, Guillaume;Vasquez-Rifo, Alejandro;Simard, Martin J.
在过去的十年里，几种新型的小非编码RNA途径被发现，它们涉及许多生物过程。所有这些途径的共同点是小RNA分子与Argonaute家族的蛋白质成员结合，形成称为RNA诱导沉默复合物或RISC的最小核心复合物。RISC以序列特异性方式靶向mRNA，通过Argonaute蛋白的内在活性诱导mRNA切割或通过仍在研究中的机制消除蛋白质合成。我们在这里详细描述了一种从秀丽隐杆线虫的提取物开始的let-7 RISC的亲和层析方法。我们的方法利用RISC的序列特异性，并利用生物素化和2 '-O-甲基化的寡核苷酸来捕获和下拉小RNA及其相关蛋白。重要的是，这种技术可以很容易地适用于靶向在不同细胞类型或模型生物中表达的其他小RNA。这种方法提供了一种有用的策略来识别与RISC相关的蛋白质，从而深入了解小RNA的功能。
During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.