lhCLIP reveals the in vivo RNA–RNA interactions recognized by hnRNPK.

RNA-RNA interactions play a crucial role in regulating gene expression and various biological processes, but identifying these interactions on a transcriptomic scale remains a challenge. To address this, we have developed a new biochemical technique called pCp-biotin labelled RNA hybrid and ultraviolet crosslinking and immunoprecipitation (lhCLIP) that enables the transcriptome-wide identification of intra- and intermolecular RNA-RNA interactions mediated by a specific RNA-binding protein (RBP). Using lhCLIP, we have uncovered a diverse landscape of intermolecular RNA interactions recognized by hnRNPK in human cells, involving all major classes of noncoding RNAs (ncRNAs) and mRNA. Notably, hnRNPK selectively binds with snRNA U4, U11, and U12, and shapes the secondary structure of these snRNAs, which may impact RNA splicing. Our study demonstrates the potential of lhCLIP as a user-friendly and widely applicable method for discovering RNA-RNA interactions mediated by a particular protein of interest and provides a valuable tool for further investigating the role of RBPs in gene expression and biological processes. Author summary: RBPs play a crucial role in post-transcriptional regulation of RNAs by modulating RNA-RNA interactions, encompassing processes like pre-mRNA splicing and mRNA degradation. Existing techniques for exploring RNA interactions associated with specific RBPs require some troublesome experimental procedures, including the use of radioisotopes to visualize RBP-RNA complexes and the introduction of exogenous expression of the target protein. In this context, we introduce lhCLIP, a novel method designed for capturing RNA-RNA interactions mediated by a specific protein. Diverging from other approaches reliant on radioisotopes, lhCLIP eliminates the need for their use. Moreover, its heightened sensitivity enables the capture of RNA-RNA interactions mediated by endogenously expressed RBPs, making it an intuitive and competitive option for researchers. We utilized lhCLIP to investigate RNA hybrids that are directly bound by hnRNPK, unveiling a complex array of intra- and intermolecular RNA interactions with the potential to exert influence over RNA splicing. [ABSTRACT FROM AUTHOR]