HITS-CLIP explained

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a variant of CLIP^[1] for genome-wide mapping protein - RNA binding sites or RNA modification sites in vivo.^{[2] [3] [4]} 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; since then a number of other splicing factor maps have been generated, including those for PTB, RbFox2,^[5] SFRS1,^[6] hnRNP C, and even N6-Methyladenosine (m6A) mRNA modifications.^{[4] [7]}

HITS-CLIP of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets^[8] by decoding microRNA-mRNA and protein-RNA interaction maps in mouse brain,^[9] and subsequently in Caenorhabditis elegans, embryonic stem cells and tissue culture cells.

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.^{[4] [7]} Recently, improved bioinformatics applied to Argonaute HITS-CLIP enables identification of binding sites with single nucleotide resolution.^[10]

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.
• iCLIP, for a thorough amplification of the cDNA library, including truncated cDNAs, thus also enabling an additional means to identify crosslink sites.

External links

• CLIPSim-MC: CLIPSim-MC is a tool that uses CLIP-seq data to find miRNA/MRE pairings using a Monte-Carlo-based approach.
• 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^[11] , 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.

Notes and References

1. Ule . Jernej . Jensen . Kirk B. . Ruggiu . Matteo . Mele . Aldo . Ule . Aljaz . Darnell . Robert B. . 2003-11-14 . CLIP identifies Nova-regulated RNA networks in the brain . Science . 302 . 5648 . 1212–1215 . 10.1126/science.1090095 . 1095-9203 . 14615540. 2003Sci...302.1212U . 23420615 .
2. Darnell RB . 2010 . HITS-CLIP: panoramic views of protein-RNA regulation in living cells . Wiley Interdiscip Rev RNA . 1 . 2. 266–86 . 10.1002/wrna.31 . 21935890 . 3222227.
3. Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB . HITS-CLIP yields genome-wide insights into brain alternative RNA processing . Nature . 456 . 7221 . 464–9 . November 2008 . 18978773 . 2597294 . 10.1038/nature07488. 2008Natur.456..464L .
4. 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. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation.. Genes & Development. 24 September 2015. 29. 19. 2037–53. 26404942. 10.1101/gad.269415.115. 4604345.
5. Yeo GW, Coufal NG, Liang TY, Peng GE, Fu XD, Gage FH . 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 . 2009 . 19136955 . 2735254 . 10.1038/nsmb.1545.
6. Sanford JR, Wang X, Mort M, Fanduyn N, Cooper DN, Mooney SD, Edenberg HJ, Liu Y . Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts . Genome Research . 19 . 3 . 381–394 . 2009 . 19116412 . 2661799 . 10.1101/gr.082503.108.
7. Ke. S. Pandya-Jones. A. Saito. Y. Fak. JJ. Vågbø. CB. Geula. S. Hanna. JH. Black. DL. Darnell. JE. Darnell. RB. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.. Genes & Development. 15 May 2017. 31. 10. 990–1006. 28637692. 10.1101/gad.301036.117. 5495127.
8. Thomson. DW . Bracken, CP . Goodall, GJ. Experimental strategies for microRNA target identification.. Nucleic Acids Research. 2011-06-07. 21652644. 10.1093/nar/gkr330. 3167600. 39. 16. 6845–6853.
9. Yang JH, Li JH, Shao P, Zhou H, Chen YQ, Qu LH . 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 . 2011 . 21037263 . 10.1093/nar/gkq1056 . 3013664.
10. Nature Biotechnology . Zhang, C. . Darnell,R.B. . Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. . 29 . 7 . 607–614 . 2011 . 21633356 . 10.1038/nbt.1873 . 3400429.
11. Predicting effective microRNA target sites in mammalian mRNAs. eLife. 2015-08-12. 2050-084X. 4532895. 26267216. e05005. 4. 10.7554/eLife.05005. en. Vikram. Agarwal. George W.. Bell. Jin-Wu. Nam. David P.. Bartel . free .