Preparation of Cardiac Extracts from Embryonal Hearts to Capture RNA- protein Interactions by CLIP

The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus.

Automated summary

The interaction between RNA and specific RNA-binding proteins plays a crucial role in gene expression regulation through complex networks. Understanding the exact environment in which this interaction occurs is important, as it can vary depending on the organism and cellular context. This study presents an optimized protocol to identify the RNA interactome of specific RNA-binding proteins, focusing on long non-coding RNAs in mouse embryonal hearts.