HITS-CLIP stands for high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, and despite what a google search will tell you, it is not a cheap player from the 90’s: those were in fact Hit-clips. HITS-CLIP utilizes in vivo UV crosslinking technologies and next-gen sequencing. CLIP differs from RIP in its use of UV crosslinking. UV light is believed to form crosslinks by exciting electrons in the bases of the RNA which then form covalent bonds with amino acids or other bases (Brimacombe et al., 1988).
This means that weak and non-specific protein interactions are not crosslinked. Since UV crosslinks are irreversible, proteinase digestion removes the protein after enrichment. A major advantage of CLIP is reduced background noise due to stringent isolation conditions made possible by crosslink stability. Further, the resolution increase of CLIP allows actual RBP binding sties to be identified down a few nucleotides. The major disadvantages of CLIP include mutations caused by UV light, inefficacy of UV crosslinking, and reduced sensitivity compared to RIP (Ascano et al., 2012).
The HITS component of HITS-CLIP was first used by (Licatalosi et al., 2008) who used it to characterize the splicing activity of the neuron specific RBP NOVO. It has since been used to generate other splicing maps (Konig et al., 2010; Xue et al., 2009; Yeo et al., 2009). iCLIP has also been applied to study microRNA-mRNA interactions by probing for Argonaute-miRNA and Argonaute-mRNA interactions and overlaying the output (Chi et al., 2009).
HITS-CLIP Additional Reading
This is an excellent review covering the HITS-CLIP protocol, data output and analysis. It also gives a historical perspective on CLIP technologies that is an excellent resource.
The mRNA binding sites of the Argonaute and Nova RBPs are mapped as proof-of-concept for the HITS-CLIP data analysis paradigm this paper presents. The authors also describe the background and principles of HITS-CLIP.
- Ascano, M., Hafner, M., Cekan, P., Gerstberger, S., and Tuschl, T. (2012). Identification of RNA-protein interaction networks using PAR-CLIP. Wiley Interdiscip. Rev. RNA 3, 159-177.
- Brimacombe, R., Stiege, W., Kyriatsoulis, A., and Maly, P. (1988). Intra-RNA and RNA-protein cross-linking techniques in Escherichia coli ribosomes. Methods Enzymol. 164, 287-309.
- Chi, S.W., Zang, J.B., Mele, A., and Darnell, R.B. (2009). Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479-486.
- Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D.J., Luscombe, N.M., and Ule, J. (2010). iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909-915.
- Licatalosi, D.D., Mele, A., Fak, J.J., Ule, J., Kayikci, M., Chi, S.W., Clark, T.A., Schweitzer, A.C., Blume, J.E., Wang, X., Darnell, J.C., and Darnell, R.B. (2008). HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464-469.
- Xue, Y., Zhou, Y., Wu, T., Zhu, T., Ji, X., Kwon, Y.S., Zhang, C., Yeo, G., Black, D.L., Sun, H., Fu, X.D., and Zhang, Y. (2009). Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Mol. Cell 36, 996-1006.
- Yeo, G.W., Coufal, N.G., Liang, T.Y., Peng, G.E., Fu, X.D., and Gage, F.H. (2009). An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130-137.