ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing.
CITATION STYLE
Carbonell, A. (2017). Immunoprecipitation and high-throughput sequencing of ARGONAUTE-bound target RNAs from plants. In Methods in Molecular Biology (Vol. 1640, pp. 93–112). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7165-7_6
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