Plant individual nucleotide resolution cross-linking and immunoprecipitation to characterize RNA-protein complexes

Abstract

In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by identification of associated RNAs by next-generation sequencing. These methods are collectively referred to as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for use in the model plant Arabidopsis thaliana.