PAR-CLIP

PAR-CLIP [1] (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) is a biochemical method for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of ribonucleoside analogs that are photoreactive, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG), into nascent RNA transcripts by living cells. Irradiation of the cells by ultraviolet light of 365 nm wavelength 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 is 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][4][5]

Similar methods

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  • CLIP-Seq, a similar method for identifying the binding sites of cellular RNA-binding proteins (RBPs) [6] or RNA modification sites [7] using UV light to cross-link RNA to RBPs without the incorporation of photoactivatable groups into RNA.

References

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  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. PMC 3156069. PMID 20644507.
  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". Nucleic Acids Res. 39 (Database issue): D202–D209. doi:10.1093/nar/gkq1056. PMC 3013664. PMID 21037263.
  4. ^ Skalsky RL, Corcoran DL, Gottwein E, Frank CL, Kang D, Hafner M, Nusbaum JD, Feederle R, Delecluse HJ, Luftig MA, Tuschl T, Ohler U, Cullen BR (2012). "The viral and cellular microRNA targetome in lymphoblastoid cell lines". PLOS Pathogens. 8 (1): e1002484. doi:10.1371/journal.ppat.1002484. PMC 3266933. PMID 22291592.
  5. ^ Gottwein E, Corcoran DL, Mukherjee N, Skalsky RL, Hafner M, Nusbaum JD, Shamulailatpam P, Love CL, Dave SS, Tuschl T, Ohler U, Cullen BR (2011). "Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines". Cell Host and Microbe. 10 (5): 515–526. doi:10.1016/j.chom.2011.09.012. PMC 3222872. PMID 22100165.
  6. ^ Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (November 2008). "HITS-CLIP yields genome-wide insights into brain alternative RNA processing". Nature. 456 (7221): 464–9. Bibcode:2008Natur.456..464L. doi:10.1038/nature07488. PMC 2597294. PMID 18978773.
  7. ^ Ke, S; Alemu, EA; Mertens, C; Gantman, EC; Fak, JJ; Mele, A; Haripal, B; Zucker-Scharff, I; Moore, MJ; Park, CY; Vågbø, CB; Kusnierczyk, A; Klungland, A; Darnell, JE; Darnell, RB (24 September 2015). "A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation". Genes & Development. 29 (19): 2037–53. doi:10.1101/gad.269415.115. PMC 4604345. PMID 26404942.
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  • starBase database: decoding miRNA-mRNA, miRNA-lncRNA, miRNA-sncRNA, miRNA-circRNA, miRNA-pseudogene, protein-lncRNA, protein-ncRNA interactions and ceRNA networks from PAR-CLIP(CLIP-Seq, HITS-CLIP,iCLIP) data, and TargetScan[1], PicTar, RNA22, miRanda and PITA microRNA target sites.
  • BIMSB doRiNA database: a database for exploring protein-RNA, microRNA-target interactions from PAR-CLIP,CLIP-Seq, HITS-CLIP,iCLIP data, and PICTAR microRNA target site predictions.
  • miRTarCLIP: A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing.
  • dCLIP: dCLIP is a Perl program for discovering differential binding regions in two comparative CLIP-Seq (HITS-CLIP, PAR-CLIP or iCLIP) experiments.
  • PARalyzer: PARalyzer is an algorithm that generates a high resolution map of interaction sites between RNA-binding proteins and their targets. The algorithm utilizes the deep sequencing reads generated from PAR-CLIP experiments.
  1. ^ Agarwal, Vikram; Bell, George W.; Nam, Jin-Wu; Bartel, David P. (2015-08-12). "Predicting effective microRNA target sites in mammalian mRNAs". eLife. 4: e05005. doi:10.7554/eLife.05005. ISSN 2050-084X. PMC 4532895. PMID 26267216.
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