Work

Protocol to study the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using cross-linking immunoprecipitation

Year: 2024

Type: article

Abstract: RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration. • Protocol to identify the direct interaction between protein and RNA in living cells • Steps to characterize the nature of nucleic acids bound to protein • Guide to characterize proteins interacting with RNA:DNA hybrids or RNA-DNA chimeras Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103292

Authors: Clara Bonnet, Ana Luisa Dian, Mélissa Leriche, Patricia Uguen, Stéphan Vagner