iCLIP

iCLIP

iCLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP. This approach includes additional steps to digest the proteins after crosslinking and to map the crosslink sites with reverse transcriptase.

In iCLIP, specific crosslinked RNA-protein complexes are immunoprecipitated. The complexes are treated with proteinase K, as the protein crosslinked at the binding site remains undigested. Upon RT, cDNA truncates at the binding site and is circularized. These circularized fragments are linearized and PCR-amplified. Deep sequencing of these amplified fragments provides nucleotide resolution of the protein-binding site eCLIP is an improvement over iCLIP that avoids circularizing the cDNA to reduce artifacts.

Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP.

Pros:

• Nucleotide resolution of protein-binding sites
• Avoids the use of nucleases
• Amplification allows the detection of rare events

Cons:

• Antibodies not specific to target will precipitate nonspecific complexes
• Nonlinear PCR amplification can lead to biases affecting reproducibility
• Artifacts may be introduced in the circularization step
• Radioisotopes required for visualization of UV-crosslinked protein-RNA complexes only label 5' ends
• Circular ligation can be inefficient

• iCLIP: Konig J., Zarnack K., Rot G., Curk T., Kayikci M., et al. (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17: 909-915

1. Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89

1. Sutandy F. X., Hildebrandt A. and Konig J. Profiling the Binding Sites of RNA-Binding Proteins with Nucleotide Resolution Using iCLIP. Methods Mol Biol. 2016;1358:175-195
2. Ennajdaoui H., Howard J. M., Sterne-Weiler T., Jahanbani F., Coyne D. J., et al. IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC. Cell Rep. 2016;15:1876-1883
3. Misra A., Ou J., Zhu L. J. and Green M. R. Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data. Genom Data. 2015;6:217-221
4. Wang Q., Taliaferro J. M., Klibaite U., Hilgers V., Shaevitz J. W., et al. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Proc Natl Acad Sci U S A. 2016;113:5269-5274
5. Gosline S. J., Gurtan A. M., JnBaptiste C. K., Bosson A., Milani P., et al. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. Cell Rep. 2016;14:310-319