Mapping of RBP-Mediated RNA-RNA Interactome with RIC-Seq

RNA-RNA interactions tend to be mediated by RNA-binding proteins (RBPs) and are involved in every step of gene regulation. Faithfully mapping RBP-mediated intra- and intermolecular RNA-RNA interactions is a prerequisite for simultaneously inferring noncoding RNA structures and targets. We present a recently developed RIC-seq (RNA in situ conformation sequencing) technology for global profiling of RBP-mediated in situ RNA-RNA interactions. Our approach fixes RBP-mediated RNA-RNA interactions in living cells with formaldehyde for subsequent permeabilization, micrococcal nuclease fragmentation, pCp-biotin labeling, and in situ RNA-RNA proximity ligation. These proximally ligated chimeric RNA fragments are subsequently enriched and converted into pair-end libraries for deep sequencing. Aligning these chimeric fragments to the genome allows the identification of the whole complement of RBP-mediated RNA-RNA interactome in cells and tissue samples. Combining these proximity information and bioinformatic tools enables the unbiased study of RNA structures and targets.