iCLIP
iCLIP
Individual nucleotide resolution CLIP (iCLIP) maps protein-RNA interactions 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 this method specific crosslinked RNA-protein complexes are immunoprecipitated. The complexes are then treated with proteinase K, as the protein crosslinked at the binding site remains undigested. Upon reverse transcription, cDNA truncates at the binding site and is circularized. These circularized fragments are then linearized and PCR-amplified. Deep sequencing of these amplified fragments provides nucleotide resolution of protein-binding site.
Pros:
- Nucleotide resolution of protein-binding site
- Avoids the use of nucleases
- Amplification allows the detection of rare events
Cons:
- Antibodies not specific to target will precipitate nonspecific complexes
- Non-linear PCR amplification can lead to biases affecting reproducibility
- Artifacts may be introduced in the circularization step
- Broughton J. P. and Pasquinelli A. E. (2013) Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP. Methods 63: 119-125
- Zarnack K., Konig J., Tajnik M., Martincorena I., Eustermann S., et al. (2013) Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152: 453-466
- Zund D., Gruber A. R., Zavolan M. and Muhlemann O. (2013) Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs. Nat Struct Mol Biol 20: 936-943
- Rogelj B., Easton L. E., Bogu G. K., Stanton L. W., Rot G., et al. (2012) Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Sci Rep 2: 603
- Tollervey J. R., Curk T., Rogelj B., Briese M., Cereda M., et al. (2011) Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci 14: 452-458