RNA-binding protein immunoprecipitation from whole-cell extracts

Abstract

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.