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

Abstract

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.Key features• The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays.• The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins.• Depending on the embryonal stage, a high number of hearts can be required as starting material.• The steps are adaptable to other tissues and biochemical assays.Graphical overviewIdentification of RNA/protein interactions from developing hearts