CLIP

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Crosslinking immunoprecipitation (CLIP) was developed in 2003[1] by the laboratory of Robert B. Darnell as a means of generating genome-wide maps of the binding sites of the Nova RNA binding protein to its RNA targets without disturbing cells (in the case of Nova, this was neurons in the brain). CLIP uses UV light to penetrate cells or tissues, inducing a covalent crosslink between proteins and nucleic acids (but not protein-protein interactions) that are within ~one Ângstrom resolution.[2] The crosslinked RNA protein complexes are then purified, typically using purification with antibodies (e.g. immunoprecipitation), the protein then removed with proteinase K, and the RNA sequenced and mapped back to the transcriptome to identify interaction sites. CLIP is in many ways analogous to DNA-ChIP (Chromatin immunoprecipitation) but provides higher resolution than typical ChIP methods that have been implemented.

One characteristic feature of CLIP is the covalent bond formed during UV irradiation, which is very specific (relative, e.g. to formaldehyde crosslinking) and allows purification of the protein-RNA complex under extremely stringent conditions. A second is that it usually incorporates partial digestion of the bound RNA, allowing fine mapping of the site of protein-RNA interaction. A third is that after protein-RNA purification, the protein can be removed with proteinase K under conditions that permit reverse-transcriptase (RT) to copy the RNA into a cDNA strand, leading to the ability to generate a PCR amplified library of RNA fragments for sequencing. The original CLIP method developed appropriate reverse transcriptase conditions to allow efficient read-through of crosslinked site and preparation of a cDNA library. Subsequently it was realized that this read-through has a higher than background (although low) error rate, and that this can be used to identify the nucleotide that is crosslinked ("cross link induced mutation site" (CIMS) analysis)[3]). Ule and colleagues also recognized that the site of cross linking induces RT to pause/arrest at sufficient frequency to generate cDNAs that are truncated at the crosslink site (iCLIP).[4]

CLIP has been used to study a wide range of proteins, with early and diverse examples including the original study of Nova in mouse brain, analysis of hnRNP A1 bound RNA in human tissue culture cells,[5] and studies in such diverse organisms as eubacteria,[6] yeast,[7] and filamentous fungus.[8] The major technologic development since the original development of CLIP was the addition of high throughput sequencing methods ("HITS-CLIP").

Related methods

  • PAR-CLIP, to identify RNA-protein interactions in tissue culture cells by using nucleotide analogs, reported by some to increase crosslinking efficiency.
  • CLIP-Seq, a synonym applied after the development of HITS-CLIP.
  • iCLIP, a modification of CLIP that takes advantage of RT pauses at sites of crosslinking

External links

  • starBase database: a database for exploring protein-RNA and miRNA-target interactions from CLIP studies.
  • clipz: a pipeline to analyze short RNA reads from HITS-CLIP experiments.

References

  1. ↑ Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB (2003), "CLIP identifies Nova-regulated RNA networks in the brain", Science 302 (5648): 1212–1215, PMID 14615540 
  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. ↑ Zhang, Chaolin; Darnell, Robert B (1 June 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. 
  4. ↑ 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 
  5. ↑ Guil S, Caceres JF (2007), "The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a", Nat Struct Mol Biol 14 (7): 591–596, PMID 17558416 
  6. ↑ Wortmann EJ, Wolin SL (2010), "A role for a bacterial ortholog of the Ro autoantigen in starvation-induced rRNA degradation", Proc Natl Acad Sci USA 107 (9): 4022–4027, doi:10.1073/pnas.1000307107, PMID 20160119 
  7. ↑ Wolf JJ, Dowell RD, Mahony S, Rabani M, Gifford DK, Fink GR. (2010), "Feed-forward regulation of a cell fate determinant by an RNA-binding protein generates asymmetry in yeast.", Genetics 185 (2): 513–522, doi:10.1534/genetics.110.113944, PMID 20382833 
  8. ↑ Becht P, König J, Feldbrügge M. (2006), "The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules", J Cell Sci 119 (Pt 23): 4964–73, PMID 17105762 
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