Insights into the design and interpretation of iCLIP experiments
journal contributionposted on 2020-08-20, 11:15 authored by Nejc Haberman, Ina Huppertz, Jan Attig, Julian König, Zhen Wang, Christian Hauer, Matthias W Hentze, Andreas E Kulozik, Hervé Le Hir, Tomaž Curk, Christopher R Sibley, Kathi Zarnack, Jernej Ule
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.
Binding site assignmentEukaryotic initiation factor 4A-III (eIF4A3)Exon-junction complexHigh-throughput sequencingPolypyrimidine tract binding protein 1 (PTBP1)Protein–RNA interactionseCLIPiCLIPirCLIPBinding SitesComputational BiologyDNA, ComplementaryExonsHeterogeneous-Nuclear RibonucleoproteinsHumansImmunoprecipitationIntronsNucleotide MotifsProtein BindingRNARNA-Binding ProteinsRibonuclease, PancreaticUltraviolet RaysUle - secBioinformatics05 Environmental Sciences06 Biological Sciences08 Information and Computing Sciences