Haberman, N;
Huppertz, I;
Attig, J;
König, J;
Wang, Z;
Hauer, C;
Hentze, MW;
... Ule, J; + view all
(2017)
Insights into the design and interpretation of iCLIP experiments.
Genome Biology
, 18
, Article 7. 10.1186/s13059-016-1130-x.
Preview |
Text (Published article)
Haberman_Insights into design.pdf - Published Version Download (2MB) | Preview |
Preview |
Text (Supplementary information)
Haberman_Insights into design_SUPPL.pdf Download (4MB) | Preview |
Abstract
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
| Type: | Article |
|---|---|
| Title: | Insights into the design and interpretation of iCLIP experiments |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1186/s13059-016-1130-x |
| Publisher version: | http://dx.doi.org/10.1186/s13059-016-1130-x |
| Language: | English |
| Additional information: | Copyright © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
| 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 |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1538467 |
Archive Staff Only
 |
View Item |
