title: Insights into the design and interpretation of iCLIP experiments.
abstract: Ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) identifies the sites on RNAs that are in direct contact with RNA-binding proteins (RBPs). Several variants of CLIP exist, which require different computational approaches for analysis. This variety of approaches can create challenges for a novice user and can hamper insights from multi-study comparisons. Here, we produce data with multiple variants of CLIP and evaluate the data with various computational methods to better understand their suitability.
  
  
  
  We perform experiments for PTBP1 and eIF4A3 using individual-nucleotide resolution CLIP (iCLIP), employing either UV-C or photoactivatable 4-thiouridine (4SU) combined with UV-A crosslinking and compare the results with published data. As previously noted, the positions of complementary DNA (cDNA)-starts depend on cDNA length in several iCLIP experiments and we now find that this is caused by constrained cDNA-ends, which can result from the sequence and structure constraints of RNA fragmentation. These constraints are overcome when fragmentation by RNase I is efficient and when a broad cDNA size range is obtained. Our study also shows that if RNase does not efficiently cut within the binding sites, the original CLIP method is less capable of identifying the longer binding sites of RBPs. In contrast, we show that a broad size range of cDNAs in iCLIP allows the cDNA-starts to efficiently delineate the complete RNA-binding sites.
  
  
  
  We demonstrate the advantage of iCLIP and related methods that can amplify cDNAs that truncate at crosslink sites and we show that computational analyses based on cDNAs-starts are appropriate for such methods.
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|The Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
authors.forename: Nejc
authors.initials: N
authors.lastname: Haberman
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.|European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Huppertz
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|The Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
authors.forename: Jan
authors.initials: J
authors.lastname: Attig
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: König
authors.affiliation: Institut de Biologie de l'ENS (IBENS), 46 rue d'Ulm, Paris, F-75005, France.|CNRS UMR 8197, Paris Cedex 05, 75230, France.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany.|Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 350, 69120, Heidelberg, Germany.|Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hauer
authors.affiliation: European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany.|Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 350, 69120, Heidelberg, Germany.
authors.forename: Matthias W
authors.initials: MW
authors.lastname: Hentze
authors.affiliation: Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 350, 69120, Heidelberg, Germany.|Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany.
authors.forename: Andreas E
authors.initials: AE
authors.lastname: Kulozik
authors.affiliation: Institut de Biologie de l'ENS (IBENS), 46 rue d'Ulm, Paris, F-75005, France.|CNRS UMR 8197, Paris Cedex 05, 75230, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Le Hir
authors.affiliation: Faculty of Computer and Information Science, University of Ljubljana, Tržaška cesta 25, 1000, Ljubljana, Slovenia.
authors.forename: Tomaž
authors.initials: T
authors.lastname: Curk
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Sibley
authors.affiliation: Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438, Frankfurt am Main, Germany. kathi.zarnack@bmls.de.
authors.forename: Kathi
authors.initials: K
authors.lastname: Zarnack
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK. j.ule@ucl.ac.uk.|The Crick Institute, 1 Midland Road, London, NW1 1AT, UK. j.ule@ucl.ac.uk.
authors.forename: Jernej
authors.identifier: 0000-0002-2452-4277
authors.initials: J
authors.lastname: Ule
mesh_terms: D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005091:Exons; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007438:Introns; D059372:Nucleotide Motifs; D011485:Protein Binding; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D018076:DNA, Complementary; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016601:RNA-Binding Proteins; D012313:RNA; D012259:Ribonuclease, Pancreatic
keywords: Binding site assignment; Eukaryotic initiation factor 4A-III (eIF4A3); Exon-junction complex; High-throughput sequencing; Polypyrimidine tract binding protein 1 (PTBP1); Protein–RNA interactions; eCLIP; iCLIP; irCLIP