iCLIP is not the latest Apple product you need to line for hours to own first, it stands for Individual-nucleotide resolution CLIP. iCLIP is a refinement of CLIP that allows single-nucleotide resolution of RBP binding sites. The protocol involves UV irradiation of living cells and immunoprecipitation of the protein of interest bound to RNAs. An adapter sequences is ligated to the 3’ ends of the RNAs.
The protein in the sample is then partially digest with proteinase K, leaving a polypeptide at the binding site. cDNA synthesis occurs from the adapter and is terminated at the polypeptide remaining on the RNA. The cDNA is then circularized meaning the nucleotide next to the adapter sequence is the last nucleotide before the RBP binding site (Huppertz et al., 2014). Single nucleotide resolution is the defining advantage of iCLIP. Further, iCLIP doesn’t need to reverse transcribe over residual amino acids like HITS-CLIP, improving accuracy.
iCLIP has no significant disadvantages; however, it requires a very detailed protocol and specialized data analysis tools to draw meaningful conclusions (Chen et al., 2014). iCLIP was introduced relatively recently in 2011 (Konig et al., 2011). Currently, iCLIP is used to characterize the splicing activity of RBPs, for example hnRNP L (Rossbach et al., 2014) and can be used to map Argonaute-miRNA binding (Broughton and Pasquinelli, 2013).
iCLIP Additional Reading
Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D.J., Luscombe, N.M., and Ule, J. (2011). iCLIP–transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. J. Vis. Exp. (50). pii: 2638. doi, 10.3791/2638.
This is the paper that introduced iCLIP, it has an additional video detailing the protocol and some of the rational for each step.
Sugimoto, Y., Konig, J., Hussain, S., Zupan, B., Curk, T., Frye, M., and Ule, J. (2012). Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions. Genome Biol. 13, R67.
This article compares discusses iCLIP in detail and compares and contrasts it to CLIP. The protocol and data analysis are compared as well as actual data sets for different RBPs.
- Broughton, J.P., and Pasquinelli, A.E. (2013). Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP. Methods 63, 119-125.
- Chen, B., Yun, J., Kim, M.S., Mendell, J.T., and Xie, Y. (2014). PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis. Genome Biol. 15, R18.
- Huppertz, I., Attig, J., D’Ambrogio, A., Easton, L.E., Sibley, C.R., Sugimoto, Y., Tajnik, M., Konig, J., and Ule, J. (2014). iCLIP: Protein-RNA interactions at nucleotide resolution. Methods 65, 274-287.
- Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D.J., Luscombe, N.M., and Ule, J. (2011). iCLIP–transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. J. Vis. Exp. (50). pii: 2638. doi, 10.3791/2638.
- Rossbach, O., Hung, L.H., Khrameeva, E., Schreiner, S., Konig, J., Curk, T., Zupan, B., Ule, J., Gelfand, M.S., and Bindereif, A. (2014). Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L. RNA Biol. 11,