PAR-CLIP

From Wikipedia, the free encyclopedia
Jump to: navigation, search

PAR-CLIP [1] (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) is a biochemical method used for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using next-generation sequencing technology.[1][2]

Recently, PAR-CLIP have been applied to determine the transcriptome-wide binding sites of several known RBPs and microRNA-containing ribonucleoprotein complexes at high resolution.[1][3]

Similar methods[edit]

  • CLIP-Seq, a similar method for identifying the binding sites of cellular RNA-binding proteins (RBPs) using UV light to cross-link RNA to RBPs without the incorporation of photoactivatable groups into RNA.

External links[edit]

References[edit]

  1. ^ a b c Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T. (2010). "Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.". Cell 141 (1): 129–141. doi:10.1016/j.cell.2010.03.009. PMC 2861495. PMID 20371350. 
  2. ^ Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M.; Jungkamp, A. C.; Munschauer, M.; Ulrich, A.; Wardle, G. S.; Dewell, S.; Zavolan, M.; Tuschl, T. (2010). "PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins". Journal of Visualized Experiments (41). doi:10.3791/2034.  edit
  3. ^ Yang JH, Li JH, Shao P, Zhou H, Chen YQ, Qu LH. (2011). "starBase: a database for exploring microRNA–mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data.". Nucl. Acids Res. 39 (Database issue): D202–D209. doi:10.1093/nar/gkq1056. PMC 3013664. PMID 21037263.