Improved binding site assignment by high-resolution mapping of RNA-protein interactions using iCLIP.

Christian Hauer,Madhuri Bhuvanagiri,Thomas Schwarzl,Matthias W Hentze,Anne-Marie Alleaume,Ina Hollerer,Wolfgang Huber,Andreas E Kulozik,Tomaz Curk,Simon Anders,Jana Sieber

doi:10.1038/ncomms8921

Abstract

Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.

Highlights

Individual-nucleotide resolution crosslinking and immunoprecipitation allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking
These methods require read-through of the reverse transcriptase (RT) beyond the crosslinking site to reach the 50 adaptor to enable amplification and deep-sequencing of the CLIP fragments. iCLIP is based on the frequent termination of the RT at the crosslinking site, which corresponds to the nucleotide preceding the start of the sequenced iCLIP fragment[6] (Fig. 1)
We show that the iCLIP libraries of a number of RBPs contain a high proportion of fragments with non-coinciding start sites, which can impact the definition of binding sites

Summary

Introduction

Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. CLIP relies on the ultraviolet light-induced formation of covalent bonds between RNA-binding proteins (RBPs) and the RNA, enabling the genome-wide and precise analysis of interactions between RNA and RBPs in vivo. This general principle underlies different protocols of CLIP analyses including HITS-CLIP2,3, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PARCLIP4,5) and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP6). We describe an analysis approach that improves the binding site assignment from such iCLIP libraries

Methods

Results

Conclusion