Progress and challenges in profiling protein–RNA and protein-associated RNA–RNA interactions

RNA binding proteins (RBPs) play essential roles in post-transcriptional gene regulation by interacting with a wide range of RNA targets. In addition to regulating RNA processing via individual RBP-RNA interactions, there is a growing appreciation of the regulatory impact of protein-associated RNA-RNA interactions that include both well-studied examples of small regulatory RNAs (e.g., microRNAs, snRNAs, snoRNAs, piRNAs) guiding ribonucleoprotein complexes to their targets as well as structured RNA elements defining the interaction landscape for an RBP. To elucidate the full scope of RBP-RNA interactions, CLIP (crosslinking and immunoprecipitation)-based methods have emerged as powerful tools. Even with the wide application of CLIP and variant approaches, these methods are still under significant ongoing advancement to better accommodate diverse biological systems and experimental demands and improve scalability. In particular, recent years have seen a focus on improved techniques to globally profile protein-associated RNA-RNA interactions. In this review, we provide a summary of recent improvements in traditional CLIP methods that improve the mapping of RBP-RNA interactions, with particular focus on those that specifically enable the profiling of protein-associated RNA-RNA interactions. We discuss the unique challenges involved in mapping protein-associated RNA-RNA interactions and highlight different ways current approaches address these challenges in order to offer a practical framework for researchers seeking to investigate RBP-associated RNA interactions.