CLIP-Seq or HITS-CLIP

High-Throughput Sequencing of CLIP cDNA Library

Crosslinking and immunoprecipitation (CLIP), with the use of RNase T1 trimming, was first described by Ule et al (Ule et al., 2005). and later applied to high-throughput sequencing to map protein-RNA binding sites in vivo (Licatalosi et al., 2008) (Chi et al., 2009). This approach is similar to RIP-Seq but uses crosslinking to stabilize the protein-RNA complexes.

In HITS-CLIP, RNA-protein complexes are UV-crosslinked and immunoprecipitated. The protein-RNA complexes are treated with RNase T1, followed by proteinase K. RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA provides single-base resolution mapping of protein binding sites on RNA.

An improvement on the HITS-CLIP protocol was published by Gillen et al., which reduces artifacts from mispriming occurences (Gillen et al., 2016).

Other versions: iCLIP, irCLIP, eCLIP, miCLIP

Advantages:

• Crosslinking stabilizes the protein-target binding
• UV crosslinking can be carried out in vivo
• Provides low background and higher resolution of binding site, due to RNase digestion
• No prior knowledge of the RNA is required
• Genome-wide RNA screen

Disadvantages:

• Over-representation of the RT complement due to mispriming (Gillen et al., 2016)
• Antibodies not specific to the target may precipitate nonspecific complexes.
• UV crosslinking is not efficient and requires close protein-RNA interactions
• Artifacts may be introduced during the crosslinking process

Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89



References:

Van Haute L., Dietmann S., Kremer L., et al. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3. Nat Commun. 2016;7:12039

Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394

Ji Z., Song R., Huang H., Regev A. and Struhl K. Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol. 2016;34:410-413

Gosline S. J., Gurtan A. M., JnBaptiste C. K., et al. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. Cell Rep. 2016;14:310-319

Nutter C. A., Jaworski E. A., Verma S. K., et al. Dysregulation of RBFOX2 Is an Early Event in Cardiac Pathogenesis of Diabetes. Cell Rep. 2016;15:2200-2213

Spengler R. M., Zhang X., Cheng C., et al. Elucidation of transcriptome-wide microRNA binding sites in human cardiac tissues by Ago2 HITS-CLIP. Nucleic Acids Res. 2016;

Gillen A. E., Yamamoto T. M., Kline E., Hesselberth J. R. and Kabos P. Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts. BMC Genomics. 2016;17:338

Moore M. J., Zhang C., Gantman E. C., Mele A., Darnell J. C. and Darnell R. B. Erratum: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc. 2016;11:616

Bennett C. G., Riemondy K., Chapnick D. A., Bunker E., Liu X., et al. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Res. 2016;44:3788-3800

Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394

Zheng Q., Bao C., Guo W., et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun. 2016;7:11215

Preusse M., Theis F. J. and Mueller N. S. miTALOS v2: Analyzing Tissue Specific microRNA Function. PLoS One. 2016;11:e0151771

Kapeli K., Pratt G. A., Vu A. Q., et al. Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses. Nat Commun. 2016;7:12143

Valentin-Vega Y. A., Wang Y. D., Parker M., et al. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation. Sci Rep. 2016;6:25996

Chen S., Blank M. F., Iyer A., et al. SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing. Nat Commun. 2016;7:10734

Zhao H., Chen M., Lind S. B. and Pettersson U. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection. Virology. 2016;492:242-250

Taliaferro J. M., Vidaki M., Oliveira R., et al. Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol Cell. 2016;61:821-833

Gaudreau M. C., Grapton D., Helness A., et al. Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells. Sci Rep. 2016;6:27379

Dugar G., Svensson S. L., Bischler T., Waldchen S., Reinhardt R., et al. The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667

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History: CLIP-Seq or HITS-CLIP

Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide