HITS-CLIP

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo.[1][2][3] HITS-CLIP was originally used to generate genome-wide protein-RNA interaction maps for the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2;[2] since then a number of other splicing factor maps have been generated, including those for PTB,[4] RbFox2,[5] SFRS1,[6] hnRNP C,[7] and even N6-Methyladenosine (m6A) mRNA modifications.[3][8]

HITS-CLIP of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets[9] by decoding microRNA-mRNA and protein-RNA interaction maps in mouse brain,[10][11] and subsequently in Caenorhabditis elegans,[12] embryonic stem cells[13] and tissue culture cells.[14]

As a novel modification of HITS-CLIP, m6A-CLIP was developed to precisely map N6-Methyladenosine(m6A) locations in mRNA by UV-crosslinking m6A antibody to the target RNA.[3][8] Recently, improved bioinformatics applied to Argonaute HITS-CLIP enables identification of binding sites with single nucleotide resolution.[15]

Similar methods
  • PAR-CLIP, for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) in tissue culture cells.
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References
  1. Darnell RB (2010). "HITS-CLIP: panoramic views of protein-RNA regulation in living cells". Wiley Interdiscip Rev RNA. 1 (2): 266–86. doi:10.1002/wrna.31. PMC 3222227. PMID 21935890.
  2. 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. doi:10.1038/nature07488. PMC 2597294. PMID 18978773.
  3. 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.
  4. Xue Y, Zhou Y, Wu T, Zhu T, Ju X, Kwon YS, Zhang C, Yeo G, Black DL, Sun H, Fu XD, 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", Molecular Cell, 36 (6): 996–1006, doi:10.1016/j.molcel.2009.12.003, PMC 2807993, PMID 20064465
  5. Yeo GW, Coufal NG, Liang TY, Peng GE, Fu XD, Gage FH (2009). "An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells". Nat Struct Mol Biol. 16 (2): 130–137. doi:10.1038/nsmb.1545. PMC 2735254. PMID 19136955.
  6. Sanford JR, Wang X, Mort M, Fanduyn N, Cooper DN, Mooney SD, Edenberg HJ, Liu Y (2009). "Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts". Genome Research. 19 (3): 381–394. doi:10.1101/gr.082503.108. PMC 2661799. PMID 19116412.
  7. Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010), "iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution", Nat Struct Mol Biol, 17 (7): 909–915, doi:10.1038/nsmb.1838, PMC 3000544, PMID 20601959
  8. Ke, S; Pandya-Jones, A; Saito, Y; Fak, JJ; Vågbø, CB; Geula, S; Hanna, JH; Black, DL; Darnell, JE; Darnell, RB (15 May 2017). "m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover". Genes & Development. 31 (10): 990–1006. doi:10.1101/gad.301036.117. PMC 5495127. PMID 28637692.
  9. Thomson, DW; Bracken, CP; Goodall, GJ (2011-06-07). "Experimental strategies for microRNA target identification". Nucleic Acids Research. 39 (16): 6845–6853. doi:10.1093/nar/gkr330. PMC 3167600. PMID 21652644.
  10. Chi, S.W.; Zang, J.B.; Mele, A.; Darnell,R.B. (2009), "Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps", Nature, 460 (7254): 479–486, doi:10.1038/nature08170, PMC 2733940, PMID 19536157
  11. 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.
  12. Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY, Pasquinelli AE, Yeo GW (2010), "Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans", Nat Struct Mol Biol, 17 (2): 173–179, doi:10.1038/nsmb.1745, PMC 2834287, PMID 20062054
  13. Leung AK, Young AG, Bhutkar A, Zheng GX, Bosson AD, Nielsen CB, Sharp PA (2011), "Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs", Nat Struct Mol Biol, 19 (9): 1084, doi:10.1038/nsmb0911-1084a, PMC 3078052
  14. 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
  15. Zhang, C.; Darnell,R.B. (2011). "Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data". Nature Biotechnology. 29 (7): 607–614. doi:10.1038/nbt.1873. PMC 3400429. PMID 21633356.
  • CLIPSim-MC: CLIPSim-MC is a tool that uses CLIP-seq data to find miRNA/MRE pairings using a Monte-Carlo-based approach.[1]
  • starBase database: a database for exploring miRNA-mRNA, miRNA-lncRNA, miRNA-sncRNA, miRNA-circRNA, miRNA-pseudogene, protein-lncRNA, protein-RNA interactions and ceRNA networks from HITS-CLIP (CLIP-Seq, PAR-CLIP, iCLIP, CLASH) data, and TargetScan[2], PicTar, RNA22, miRanda and PITA microRNA target sites.
  • clipz: a pipeline to analyze short RNA reads from HITS-CLIP experiments.
  • dCLIP: dCLIP is a Perl program for discovering differential binding regions in two comparative CLIP-Seq (HITS-CLIP, PAR-CLIP or iCLIP) experiments.
  1. Peter M. Clark; Phillipe Loher; Kevin Quann; Jonathan Brody; Eric R. Londin; Isidore Rigoutsos (2014), "Argonaute CLIP-Seq reveals miRNA targetome diversity across tissue types", Scientific Reports, 4 (5947): 5947, doi:10.1038/srep05947, PMC 4894423, PMID 25103560
  2. 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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