Vector-borne Virus Research Center, State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China.
Methods Mol Biol. 2022;2400:139-147. doi: 10.1007/978-1-0716-1835-6_14.
Argonaute (AGO) proteins associate with small RNAs (sRNAs) to form an RNA-induced silencing complex (RISC). The ribonuclease (slicer) activity of AGOs is required for the sRNA-complementary target cleavage, which is important for RISC-mediated RNA silencing, especially in plants. Sequencing small RNAs is an obvious choice to understand their expression and downstream effects. It also provides an opportunity to identify novel and polymorphic miRNAs. Recently, we have successfully reconstituted rice (Oryza sativa) AGO1a slicer assays in vitro that are able to recapitulate in vivo miRNA-guided cleavage activity. Here we provide a detailed protocol for the purification of OsAGO1a-sRNA complexes and further slicer assays, small RNA sequencing and bioinformatic analysis. This protocol can be readily adapted for the purification and subsequent analyses of the AGO complexes in other plants.
Argonaute (AGO) 蛋白与小 RNA (sRNA) 结合形成 RNA 诱导沉默复合物 (RISC)。AGO 的核糖核酸酶 (slicer) 活性对于 sRNA 互补靶标切割是必需的，这对于 RISC 介导的 RNA 沉默非常重要，尤其是在植物中。测序小 RNA 是了解其表达和下游效应的明显选择。它还提供了识别新的和多态性 miRNA 的机会。最近，我们已经成功地在体外重建了水稻 (Oryza sativa) AGO1a 核酸内切酶测定，该测定能够重现体内 miRNA 指导的切割活性。在这里，我们提供了一个详细的方案，用于纯化 OsAGO1a-sRNA 复合物，并进一步进行核酸内切酶测定、小 RNA 测序和生物信息学分析。该方案可以很容易地适应于其他植物中 AGO 复合物的纯化和后续分析。
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