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Insights into the design and interpretation of iCLIP experiments.
- Source :
-
Genome biology [Genome Biol] 2017 Jan 16; Vol. 18 (1), pp. 7. Date of Electronic Publication: 2017 Jan 16. - Publication Year :
- 2017
-
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.<br />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.<br />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.
- Subjects :
- Binding Sites
Computational Biology methods
DNA, Complementary genetics
DNA, Complementary metabolism
Exons
Heterogeneous-Nuclear Ribonucleoproteins metabolism
Humans
Introns
Nucleotide Motifs
Protein Binding
Ribonuclease, Pancreatic metabolism
Ultraviolet Rays
Immunoprecipitation methods
RNA genetics
RNA metabolism
RNA-Binding Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1474-760X
- Volume :
- 18
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Genome biology
- Publication Type :
- Academic Journal
- Accession number :
- 28093074
- Full Text :
- https://doi.org/10.1186/s13059-016-1130-x