One of the challenges to assess the impact of RBPs on posttranscriptional gene regulation is identifying and quantifying the RNA and protein components of ribonucleoprotein complexes (RNPs) in a cellular context. The human genome is estimated to contain 1542 RBPs. Recent studies indicated that approximately 800 proteins are bound to polyadenylated RNA within a single human cell type. Early approaches to identify RBP targets en masse independent of physiological context involved in vitro selection against limited RNA libraries. The onset of microarray technology allowed for assessing RBP binding to RNA in a genome-wide manner. In these assays, RNP complexes are immunoprecipitated (RIP) from cell lysates. Associated RNAs are then isolated from these RNP complexes and interrogated with either microarray (RIP-chip) or sequencing (RIP-seq) technology. Unlike UV cross-linking and immunoprecipitation (CLIP-seq) methods, RIP-seq allows for the detection of RNA components of RNPs that are not directly bound to the RBP of interest. This is particularly relevant when interrogating multicomponent RNPs such as the exon junction complex. RIP-seq provides whole transcript-level binding information rather than the site-level resolution of CLIP-seq methods. Comparisons have shown that RNAs with more CLIP-defined binding sites are more likely to be enriched in the RIP, suggesting that RIP identifies the more stably associated RNAs.
Expected outcome: Enrichment of ACTB in FLAG IP relative to IgG IP and input. IGV browser snapshot of RIP-seq data for ACTB depicts normalized RIP-seq coverage (red) for the FLAG IP (top), IgG IP (middle), and input RNA (bottom). PAR-CLIP- defined binding sites (blue) of ELAVL/HuR from 4 independent libraries all performed in HEK293 cells.