RNA-binding protein immunoprecipitation from whole-cell extracts.
RNA-based regulation is increasingly recognized as an important factor shaping the cellular transcriptome. RNA-binding proteins that interact with cis-regulatory motifs within pre-mRNAs determine the fate of their targets. Understanding posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe RNA immunoprecipitation in Arabidopsis thaliana. Transgenic plants expressing an RNA-binding protein fused to green fluorescent protein are treated with formaldehyde to "trap" RNAs in complexes with their physiological protein partners. A whole-cell extract is subjected to immunoprecipitation with an antibody against the GFP tag. In parallel, a mock immunoprecipitation is performed using an unrelated antibody. Coprecipitated RNAs are eluted from the immunoprecipitate and identified via real-time PCR. Enrichment relative to immunoprecipitation from plants expressing GFP only and mock immunoprecipitation with an unrelated antibody indicates specific binding.
Department of Molecular Cell Physiology, Institute for Genome Research and Systems Biology, University of Bielefeld, Bielefeld, Germany.
Tino
T
Köster
Dorothee
D
Staiger
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D047468:Immunoprecipitation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins