CLIP (cross-linking immunoprecipitation) is a method used in molecular biology that combines UV cross-linking with immunoprecipitation in order to analyse protein interactions with RNA. CLIP-based techniques can be used to map RNA binding sites for a protein of interest on a genome-wide scale, thereby increasing the understanding of post-transcriptional regulatory networks.
CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV). Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity. The cross-linked cells are then lysed, and the protein of interest is isolated via immunoprecipitation. In order to allow for sequence specific priming of reverse transcription, RNA adapters are ligated to the 3' ends, while radiolabeled phosphates are transferred to the 5' ends of the RNA fragments. The RNA-protein complexes are then separated from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the RNA-protein complexes. This step leaves a peptide at the cross-link site, allowing for the identification of the cross-linked nucleotide. After ligating RNA linkers to the RNA 5' ends, cDNA is synthesized via RT-PCR. High-throughput sequencing is then used to generate reads containing distinct barcodes that identify the last cDNA nucleotide. Interaction sites can be identified by mapping the reads back to the transcriptome.
History and applications
CLIP was originally undertaken to study interactions between the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2 in the mouse brain, identifying RNA binding sites that had Nova binding sites and were validated as Nova targets in knock-out mouse brains. In 2008 CLIP was combined with high-throughput sequencing (termed "HITS-CLIP") to generate genome-wide protein-RNA interaction maps for Nova; since then a number of other splicing factor maps have been generated, including those for PTB, RbFox2 (where it was renamed "CLIP-seq") SFRS1, Argonaute, hnRNP C, the Fragile-X mental retardation protein FMRP, Ptbp2 (in the mouse brain) Mbnl2, and the nElavl proteins (the neuron-specific Hu proteins). A review of the range of proteins studied by HITS-CLIP has been published.
HITS-CLIP (CLIP-seq) analysis of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets by decoding microRNA-mRNA and protein-RNA interaction maps in the mouse brain, and subsequently in Caenorhabditis elegans, embryonic stem cells and tissue culture cells. Recently, improved bioinformatics methods applied to Argonaute HITS-CLIP, have identified binding sites with single nucleotide resolution.
miRNA target detection
The main steps (using Degradome sequencing concurrently) are:
- mapping CLIP-seq reads
- mapping Degradome-Seq reads
- grouping overlapping reads into clusters
- querying miRNA targets from different public databases
- identifying 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
HITS-CLIP or CLIP-Seq
HITS-CLIP also known as CLIP-Seq combines UV cross-linking and immunoprecipitation with high-throughput sequencing to identify binding sites of RNA-binding proteins. CLIP-seq depends on cross-linking induced mutation sites (CIMS) to localized protein-RNA binding sites. Because CIMS are reproducible, high sequencing depths allow CIMS to be differentiated from technical errors.
PAR-CLIP  (Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation) is a biochemical method used for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient cross-linking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by the isolation of the cross-linked and co-immunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using high-throughput sequencing technology. Cross-linking the 4-SU and 6-SG analogs results in thymidine to cytidine, and guanosine to adenosine transitions respectively. As a result, PAR-CLIP can identify binding site locations with high accuracy.
However, PAR-CLIP is limited to cultured cells, and nucleoside cytotoxicity is a concern; it has been reported that 4-SU inhibits ribosomal RNA synthesis, induces a nucleolar stress response, and reduces cell proliferation. It should be noted that 4-SU substitution occurs in approximately 1 out of every 40 uridine nucleosides, and that T to C transitions frequently occur at the cross-link site.
Recently, PAR-CLIP has been employed to determine the transcriptome-wide binding sites of several known RBPs and microRNA-containing ribonucleoprotein complexes at high resolution. This includes the miRNA targeting AGO and TNRC6 proteins.
iCLIP (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation) is a technique used for identifying protein-RNA interactions. The method uses UV light to covalently bind proteins and RNA molecules. As with all CLIP methods, iCLIP allows for the stringent purification of linked protein-RNA complexes, using immunoprecipitation followed by SDS-PAGE and membrane transfer. The radiolabelled protein-RNA complexes are then excised from the membrane, and treated with proteinase to release the RNA. This leaves one or two amino acids at the RNA cross-link site. The RNA is then reverse transcribed using barcoded primers. Because reverse transcription stops prematurely at the cross-link site, iCLIP allows RNA-protein interaction sites to be identified at high resolution.
CLIP advantages and limitations
Early methods for identifying RNA-protein interactions relied on either the affinity purification of RNA-binding proteins or the immunoprecipitaiton of RNA-protein complexes. These methods lacked a cross-linking step and obtained low signal to noise ratios. Because RNA-binding proteins are frequently components of multi-protein complexes, RNAs bound to non-target proteins may be co-precipitated. The data obtained using early immunoprecipitation methods have been demonstrated to be dependent on the reaction conditions of the experiment. For example, the subset of RNA-protein interactions preserved depends highly on the protein concentrations and ionic conditions. Furthermore, the reassociation of RNA-binding proteins following cell lysis may lead to the detection of artificial interactions.
Formaldehyde cross-linking methods have been used to preserve RNA-protein interactions, but also generate protein-protein cross-links. UV cross-linking methods provide a significant advantage over formaldehyde cross-linking, as they avoid protein-protein cross-links entirely. Proteinase K digestion also confers an advantage to CLIP methods due to the peptide left at the cross-link site. Reverse transcription of the fragments through the cross-link site introduces mutations that are specific to each separate CLIP method, and may be used to determine the binding site with high accuracy.)
All CLIP library generation protocols require moderate quantities of cells or tissue (50–100 mg), require numerous enzymatic steps, and, for HITS-CLIP, extensive informatic analysis (as recently reviewed). Certain steps are difficult to optimize and frequently have low efficiencies. For example, overdigestion with RNase can decrease the number of identified binding sites. Cross-linking also presents a concern. The optimal cross-linking protocol varies between proteins, and the efficiency is typically between 1-5%. Cross-linking bias has been reported in the literature, but the impact of biases present in CLIP methods remains debatable. Because CLIP methods rely on immunoprecipitation, antibody-epitope interactions are a potential obstacle. For instance, cross-linking at the epitope could impede antibody binding. Finally, significant differences have been observed between cross-linking sites in vivo in living cells and in vitro. Therefore, CLIP results may not necessarily reflect RNA-protein binding site interactions within the cell.
- RIP-Chip, same goal and first steps, but doesn't use cross-linking 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
- Ule, J; Jensen, K; Ruggiu, M; Mele, A; Ule, A; Darnell, RB (Nov 14, 2003). "CLIP identified Nova-regulated RNA networks in the brain.". Science 302 (5648): 1212–1215. doi:10.1126/science.1090095. PMID 14615540.
- Sugimoto, Y; König, J; Hussain, S; Zupan, B; Curk, T; Frye, M; Ule, J (Aug 3, 2012). "Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions.". Genome Biology 13 (8): R67. doi:10.1186/gb-2012-13-8-r67. PMC 4053741. PMID 22863408.
- 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. PMC 3400429. PMID 21633356.
- Darnell, R. (2012). "CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein". Cold Spring Harbor Protocols 2012 (11): pdb.prot072132. doi:10.1101/pdb.prot072132.
- König, J.; McGlincy, N. J.; Ule, J. (2012). "Analysis of Protein-RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing". Tag-Based Next Generation Sequencing. p. 153. doi:10.1002/9783527644582.ch10. ISBN 9783527644582.
- Ule J, Jensen K, Mele A, Darnell RB. (December 2005). "CLIP: a method for identifying protein-RNA interactions sites in living cells". Methods 37 (4): 376–386. doi:10.1016/j.ymeth.2005.07.018. PMID 16314267.
- 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.
- 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, PMID 20064465
- 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.
- 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.
- Chi SW, Zang JB, Mele A, Darnell RB (2009). "Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps". Nature 460 (7254): 479–486. doi:10.1038/nature08170. PMC 2733940. PMID 19536157.
- 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
- Darnell JC, Van Driesche SJ, Zhang C, Hung KY, Mele A, Fraser CE, Stone EF, Chen C, Fak JJ, Chi SW, Licatalosi DD, Richter JD, Darnell RB (2011). "FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.". Cell 146 (2): 247–261. doi:10.1016/j.cell.2011.06.013. PMC 3232425. PMID 21784246.
- Licatalosi DD, Yano M, Fak JJ, Mele A, Grabinski SE, Zhang C, Darnell RB (2012). "Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain". Genes Dev 26 (14): 1626–1642. doi:10.1101/gad.191338.112. PMC 3404389. PMID 22802532.
- Charizanis K, Lee KY, Batra R, Goodwin M, Zhang C, Yuan Y, Shiue L, Cline M, Scotti MM, Xia G, Kumar A, Ashizawa T, Clark HB, Kimura T, Takahashi MP, Fujimura H, Jinnai K, Yoshikawa H, Gomes-Pereira M, Gourdon G, Sakai N, Nishino S, Foster TC, Ares M Jr, Darnell RB, Swanson MS (2012). "Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy.". Neuron 75 (3): 437–450. doi:10.1016/j.neuron.2012.05.029. PMC 3418517. PMID 22884328.
- Ince-Dunn G, Okano HJ, Jensen KB, Park WY, Zhong R, Ule J, Mele A, Fak JJ, Yang C, Zhang C, Yoo J, Herre M, Okano H, Noebels JL, Darnell RB (2012). "Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability.". Neuron 75 (6): 1067–1080. doi:10.1016/j.neuron.2012.07.009. PMC 3517991. PMID 22998874.
- Darnell RB (2010). "HITS-CLIP: panoramic views of protein-RNA regulation in living cells.". Wiley Interdiscip Rev RNA 1 (2): 266–286. doi:10.1002/wrna.31. PMC 3222227. PMID 21935890.
- Thomson, DW; Bracken, CP, Goodall, GJ (2011-06-07). "Experimental strategies for microRNA target identification.". Nucleic Acids Research. doi:10.1093/nar/gkr330. PMC 3167600. PMID 21652644.
- 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
- 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.
- 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
- 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, PMID 21894221
- 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
- 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. PMC 3222227. PMID 21935890.
- 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. PMC 3400429. PMID 21633356.
- König, J; Zarnack, K; Luscombe, NM; Ule, J (Jan 18, 2012). "Protein-RNA interactions: new genomic technologies and perspectives.". Nature reviews. Genetics 13 (2): 77–83. doi:10.1038/nrg3141. PMID 22251872.
- 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.
- 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.
- Burger K, Mühl B, Kellner M, Rohrmoser M, Gruber-Eber A, Windhager L, Friedel CC, Dölken L, Eick D. (2013). "4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response.". RNA Biol 10 (10): 1623–1630. doi:10.4161/rna.26214. PMID 24025460.
- Ule J, Jensen K, Mele A, Darnell RB (2005). "CLIP: a method for identifying protein-RNA interaction sites in living cells.". Methods 37 (4): 376–86. doi:10.1016/j.ymeth.2005.07.018. PMID 16314267.
- Mili, S; Steitz, J. A. (2004). "Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses". RNA 10 (11): 1692–4. doi:10.1261/rna.7151404. PMC 1370654. PMID 15388877.
- Moore JJ, Zhang C, Gantman EC, Mele A, Darnell JC, Darnell RB (2014), "Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.", Nat Protocols 9 (2): 263–293, PMID 24407355
- König, J; Zarnack, K; Luscombe, N. M.; Ule, J (2012). "Protein-RNA interactions: New genomic technologies and perspectives". Nature Reviews Genetics 13 (2): 77–83. doi:10.1038/nrg3141. PMID 22251872.
- Ule, J; Jensen, K; Mele, A; Darnell, R. B. (2005). "CLIP: A method for identifying protein-RNA interaction sites in living cells". Methods 37 (4): 376–86. doi:10.1016/j.ymeth.2005.07.018. PMID 16314267.
- Fecko, C. J.; Munson, K. M.; Saunders, A; Sun, G; Begley, T. P.; Lis, J. T.; Webb, W. W. (2007). "Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking". Photochemistry and Photobiology 83 (6): 1394–404. doi:10.1111/j.1751-1097.2007.00179.x. PMID 18028214.
- Bohnsack, M. T.; Martin, R; Granneman, S; Ruprecht, M; Schleiff, E; Tollervey, D (2009). "Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis". Molecular Cell 36 (4): 583–92. doi:10.1016/j.molcel.2009.09.039. PMC 2806949. PMID 19941819.
- 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.
- 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.