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
Related
History: CLIP-Seq or HITS-CLIP
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
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 SelectorReviews:Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89References: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;7:12039Vourekas 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;531:390-394Ji Z., Song R., Huang H., Regev A. and Struhl K. Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol. 2016;34:410-413Gosline 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;14:310-319Nutter 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;15:2200-2213Spengler 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;17:338Moore 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;11:616Bennett 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;44:3788-3800Vourekas 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;531:390-394Zheng 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;7:11215Preusse M., Theis F. J. and Mueller N. S. miTALOS v2: Analyzing Tissue Specific microRNA Function. PLoS One. 2016;11:e0151771Kapeli 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;7:12143Valentin-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;6:25996Chen S., Blank M. F., Iyer A., et al. SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing. Nat Commun. 2016;7:10734Zhao 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;492:242-250Taliaferro J. M., Vidaki M., Oliveira R., et al. Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol Cell. 2016;61:821-833Gaudreau 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;6:27379Dugar 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