Rapid and Specific Purification of Argonaute-Small RNA Complexes from Rice for Slicer Activity

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.