PAR-CLIP
Par-CLIP
Photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation (PAR-CLIP) maps RNA-binding proteins (RBPs) . This approach is similar to HITS-CLIP and CLIP-Seq, but uses much more efficient crosslinking to stabilize the protein-RNA complexes. The requirement to introduce a photoactivatable ribonucleoside limits this approach to cell culture and in vitro systems. In this method, 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) are incorporated into transcripts of cultured cells. UV irradiation crosslinks 4-SU/6-SG–labeled transcripts to interacting RBPs. The targeted complexes are immunoprecipitated and digested with RNase T1, followed by Proteinase K, before RNA extraction. The RNA is reverse-transcribed to cDNA and sequenced. Deep sequencing of cDNA accurately maps RBPs interacting with labeled transcripts.
Pros:
- Highly accurate mapping of RNA-protein interactions
- Labeling with 4-SU/6-SG improves cross-linking efficiency
Cons:
- Antibodies not specific to target may precipitate nonspecific complexes
- Limited to cell culture and in vitro systems
- Chen T., Hao Y. J., Zhang Y., Li M. M., Wang M., et al. (2015) m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell 16: 289-301
- Conn S. J., Pillman K. A., Toubia J., Conn V. M., Salmanidis M., et al. (2015) The RNA binding protein quaking regulates formation of circRNAs. Cell 160: 1125-1134