Protein Binding to mRNA 3′ Isoforms

Abstract

Here we describe CLIP-READS, a technique that combines elements of crosslinking and immunoprecipitation (CLIP) and 3′ region extraction and deep sequencing (READS), to provide a genome-wide map of mRNA 3′ isoform binding by a given messenger ribonucleoprotein (mRNP). In CLIP-READS, cells are grown to logarithmic phase and are irradiated with UV light (254 nm) to form RNA–protein adducts. The protein−mRNA complexes are immunoprecipitated from cell extracts with an antibody specific to the protein of interest, after which the protein component is digested away with Pronase. Messenger RNAs are then subjected to 3′ READS. An input sample processed by 3′ READS in parallel allows for the relative quantification of isoform-specific binding by the mRNP of interest. © 2019 by John Wiley & Sons, Inc.