Decoding RNA–protein interactions using high-throughput methods

RNA-binding proteins (RBPs) constitute a diverse class of proteins essential for every stage of the gene expression process. Many RBPs are also linked to human diseases and pathologies. Understanding the molecular grammar of RNA-protein interactions is critical for deciphering the regulatory RNA code. This review provides a comprehensive overview of Massively Parallel Binding Assays (MPBAs), high-throughput techniques that use large libraries of RNA or protein variants to systematically investigate RNA-protein interactions. We describe the underlying principles of both in vitro and in vivo approaches, their applications, as well as their strengths and weaknesses. We conclude by outlining future directions and challenges in the field that will help drive the development of novel methods to better understand the RBP recognition code.