CLIP-Seq

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CLIP-Seq, short for cross-linking immunoprecipitation-high-throughput sequencing[1] less commonly called HITS-CLIP,[2][3] is a method in molecular biology used for screening for RNA sequences that interact with a particular RNA-binding protein (or another RNA[4]). It employs UV-crosslinking between RNA and the protein, followed by immunoprecipitation with antibodies for the protein, fragmentation, high-throughput sequencing and bioinformatics.

HITS-CLIP (CLIP-Seq) of Argonaute has been applied to decode microRNA-target interaction maps.[5][6][7] The application of CLIP-Seq/HITS-CLIP methods has significantly reduced the rate of false positive predictions of miRNA binding sites and has also reduced the size of the search space for miRNA target sites.[5][6][7] Improved analysis of Argonaute HITS-CLIP now enables identification of binding sites with single nucleotide resolution.[8]


miRNA targets detection


The main steps (using at the same time degradome data Degradome sequencing) are:

  • mapping CLIP-seq reads
  • mapping Degradome-Seq reads
  • the overlapping reads were grouped into clusters
  • miRNA targets from different public databases
  • miRNA–target interactions with an alignment score from CleaveLand not exceeding the cutoff threshold of 7.0
  • the ClipSearch program was developed to search for 6–8-mers (8-mer, 7-mer-m8 and 7-mer-A1) (2,5) in CLIP-Seq data
  • The DegradomeSearch program was developed to search Degradome-Seq clusters for nearly perfect complements of miRNA sequences

Similar methods

  • PAR-CLIP, another method for identifying the binding sites of cellular RNA-binding proteins (RBPs) also including microRNA-containing ribonucleoprotein complexes (miRNPs) in tissue culture cells.
  • RIP-Chip, same goal and first steps, but doesn't use crosslinking and uses microarray instead of sequencing
  • ChIP-Seq, for finding interactions with DNA rather than RNA
  • SELEX, a method for finding a consensus binding sequence

External links

  • starBase database: a database for exploring miRNA-lncRNA, miRNA-mRNA, miRNA-sncRNA, miRNA-circRNA, protein-lncRNA, protein-RNA interactions and ceRNA networks from PAR-CLIP(CLIP-Seq, HITS-CLIP,iCLIP, CLASH) data, and TargetScan, PicTar, RNA22, miRanda and PITA microRNA target sites.
  • BIMSB doRiNA database: a database for exploring protein-RNA and microRNA-target interactions from CLIP-Seq, HITS-CLIP, PAR-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.

References

  1. ↑ Sanford JR, Wang X, Mort M, et al. (March 2009). "Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts". Genome Res. 19 (3): 381–94. doi:10.1101/gr.082503.108. PMC 2661799. PMID 19116412. 
  2. ↑ Darnell RB (2010). "HITS-CLIP: panoramic views of protein-RNA regulation in living cells". Wiley Interdiscip Rev RNA. 1: 266–86. doi:10.1002/wrna.31. 
  3. ↑ Licatalosi DD, Mele A, Fak JJ, et al. (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. 
  4. ↑ Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS (April 2009). "Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling". Science 324 (5924): 218–23. doi:10.1126/science.1168978. PMC 2746483. PMID 19213877. 
  5. ↑ 5.0 5.1 Chi,S.W., Zang,J.B., Mele,A. and 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. 
  6. ↑ 6.0 6.1 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. 
  7. ↑ 7.0 7.1 Zisoulis,D.G., Lovci,M.T., Wilbert,M.L., Hutt,K.R., Liang,T.Y., Pasquinelli,A.E. and Yeo,G.W. (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. 
  8. ↑ Zhang,C. and 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. PMID 21633356. 


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