使用 iCLIP 鉴定秀丽隐杆线虫中的 Argonaute 结合位点。
Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP.
机构信息
Division of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA.
出版信息
Methods. 2013 Sep 15;63(2):119-25. doi: 10.1016/j.ymeth.2013.03.033. Epub 2013 Apr 10.
Abstract
The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. Past approaches using in silico methods and reporter constructs lack biological context that may enhance or inhibit target recognition. To address these limitations, several labs have utilized crosslinking and immunoprecipitation (CLIP) of Argonaute (Ago) proteins to identify miRNA targets. Recently, the Ule Lab introduced individual-nucleotide resolution CLIP (iCLIP) to increase the sensitivity of identifying protein-RNA interaction sites. Here we adapt the iCLIP protocol for use in Caenorhabditis elegans to identify endogenous sites targeted by the worm Argonaute (ALG-1) primarily responsible for miRNA function.
摘要
内源性靶标的鉴定仍然是理解 microRNA（miRNA）功能的一个重要挑战。过去使用计算机方法和报告基因构建体的方法缺乏可能增强或抑制靶标识别的生物学背景。为了解决这些限制，几个实验室利用 Argonaute（AGO）蛋白的交联和免疫沉淀（CLIP）来鉴定 miRNA 靶标。最近，Ule 实验室引入了单个核苷酸分辨率 CLIP（iCLIP）来提高鉴定蛋白-RNA 相互作用位点的灵敏度。在这里，我们改编了 iCLIP 方案，用于鉴定线虫 Argonaute（ALG-1）的内源性靶标，该蛋白主要负责 miRNA 的功能。
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