Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) attempts to solve some of the problems of HITS-CLIP, namely, efficiency of crosslinking and resolution of RBP binding sites. The unique aspect of PAR-CLIP is the incorporation of photoreactive ribonucleoside analogs into nascent RNA of living cells.
These analogs facilitate highly efficient crosslinking of RNA to proteins. The next step is typically CLIP immunoprecipitation of the RBP followed by cDNA synthesis and detection via sequencing. When the photoreactive analog undergoes a crosslinking, it appears as a characteristic mutation in the sequence (Spitzer et al., 2014). This allows for the actual binding sites of the RBP to be detected. PAR-CLIP therefore has increased resolution and decreased signal-to-noise ratio compared to other technologies. The major issue with PAR-CLIP is that not all experimental systems are conducive to incorporation of the ribonucleoside analogs, only cell culture is appropriate for animal studies.
PAR-CLIP was introduced in 2010 where it was used to to identify the recognition sequence motifs of several important miRNA binding proteins (Hafner et al., 2010). Since, databases containing many PAR-CLIP data sets have been made publicly available and allow in silico discovery of novel interactions, such as miRNA-lncRNA (Jalali et al., 2013).
PAR–CLIP Additional Reading
The rational and background for the development of each step of PAR-CLIP compared to other CLIP protocols are presented in this review. The authors discuss different crosslinking methods, ribonucleoside analog biochemistry, and challenges in identification of RBPs.
- Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano, M.,Jr, Jungkamp, A.C., Munschauer, M., et al. (2010). Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129-141.
- Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano, M., Jungkamp, A.C., Munschauer, M., et al. (2010). PAR-CliP–a method to identify transcriptome-wide the binding sites of RNA binding proteins. J. Vis. Exp. (41). pii: 2034. doi, 10.3791/2034.
- Jalali, S., Bhartiya, D., Lalwani, M.K., Sivasubbu, S., and Scaria, V. (2013). Systematic transcriptome wide analysis of lncRNA-miRNA interactions. PLoS One 8, e53823.
- Spitzer, J., Hafner, M., Landthaler, M., Ascano, M., Farazi, T., Wardle, G., Nusbaum, J., Khorshid, M., Burger, L., Zavolan, M., and Tuschl, T. (2014). PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins. Methods Enzymol. 539, 113-161.