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

Christian Hauer, Tomaz Curk, Simon Anders, Thomas Schwarzl, Anne-Marie Alleaume, Jana Sieber, Ina Hollerer, Madhuri Bhuvanagiri, Wolfgang Huber, Matthias W. Hentze () and Andreas E. Kulozik ()
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Christian Hauer: Hematology and Immunology, University of Heidelberg
Tomaz Curk: European Molecular Biology Laboratory (EMBL) Heidelberg
Simon Anders: European Molecular Biology Laboratory (EMBL) Heidelberg
Thomas Schwarzl: European Molecular Biology Laboratory (EMBL) Heidelberg
Anne-Marie Alleaume: European Molecular Biology Laboratory (EMBL) Heidelberg
Jana Sieber: Hematology and Immunology, University of Heidelberg
Ina Hollerer: Hematology and Immunology, University of Heidelberg
Madhuri Bhuvanagiri: Hematology and Immunology, University of Heidelberg
Wolfgang Huber: European Molecular Biology Laboratory (EMBL) Heidelberg
Matthias W. Hentze: Molecular Medicine Partnership Unit (MMPU)
Andreas E. Kulozik: Hematology and Immunology, University of Heidelberg

Nature Communications, 2015, vol. 6, issue 1, 1-13

Abstract: 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.

Date: 2015
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DOI: 10.1038/ncomms8921

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