PAR-CLIP
Photoactivatable Ribonucleoside_Enhanced Crosslinking and Immunoprecipitation
PAR-CLIP maps RBP sites on the target RNAs (Hafner et al., 2008). This approach is similar to HITS-CLIP and CLIP-Seq, but it uses much more efficient crosslinking to stabilize the protein-RNA complexes. The requirement to introduce a photoactivatable ribonucleoside limits this approach to cell culture and in vitro systems.
In this method, 4-SU and 6-thioguanosine (6-SG) are incorporated into transcripts of cultured cells. UV irradiation crosslinks 4-SU/6-SG_labeled transcripts to interacting RBPs. The targeted complexes are immunoprecipitated and digested with RNase T1, followed by proteinase K, before RNA extraction. The RNA is reverse-transcribed to cDNA and sequenced. Deep sequencing of cDNA accurately maps RBPs interacting with labeled transcripts.
Advantages:
- Highly accurate mapping of RNA-protein interactions
- Labeling with 4-SU/6-SG improves crosslinking efficiency
- Legitimately crosslinked sequences can be identified based on the presence of mutated bases, and mispriming can be filtered bioinformatically (Gillen et al., 2016)
Disadvantages:
- Antibodies not specific to the target may precipitate nonspecific complexes
- Limited to cell culture and in vitro systems
- Photoreactive nucleosides can be cytotoxic (Kloetgen et al., 2015)
- High concentrations of 4-SU can inhibit rRNA synthesis and induce a nucleolar stress response (Burger et al., 2013)
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
Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236
References:
Tichon A., Gil N., Lubelsky Y., Havkin Solomon T., Lemze D., et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat Commun. 2016;7:12209
Vongrad V., Imig J., Mohammadi P., et al. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages. PLoS One. 2015;10:e0132127
Liu N., Dai Q., Zheng G., He C., Parisien M. and Pan T. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015;518:560-564
Murakawa Y., Hinz M., Mothes J., et al. RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKKF-kappaB pathway. Nat Commun. 2015;6:7367
Kang D., Skalsky R. L. and Cullen B. R. EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival. PLoS Pathog. 2015;11:e1004979
Matveeva E., Maiorano J., Zhang Q., et al. Involvement of PARP1 in the regulation of alternative splicing. Cell Discov. 2016;2:15046
Porter D. F., Koh Y. Y., VanVeller B., Raines R. T. and Wickens M. Target selection by natural and redesigned PUF proteins. Proc Natl Acad Sci U S A. 2015;112:15868-15873
Wang Q., Taliaferro J. M., Klibaite U., Hilgers V., Shaevitz J. W. and Rio D. C. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Proc Natl Acad Sci U S A. 2016;113:5269-5274
Lee A. S., Kranzusch P. J. and Cate J. H. eIF3 targets cell-proliferation messenger RNAs for translational activation or repression. Nature. 2015;522:111-114
Chu Y., Wang T., Dodd D., Xie Y., Janowski B. A. and Corey D. R. Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells. Nucleic Acids Res. 2015;43:e75
Xie H., Lee L., Scicluna P., et al. Novel functions and targets of miR-944 in human cervical cancer cells. Int J Cancer. 2015;136:E230-241
Xiong X. P., Vogler G., Kurthkoti K., Samsonova A. and Zhou R. SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function. PLoS Genet. 2015;11:e1005475
Chen T., Hao Y. J., Zhang Y., et al. m(6)A RNA Methylation Is Regulated by MicroRNAs and Promotes Reprogramming to Pluripotency. Cell Stem Cell. 2015;16:289-301
Related
History: PAR-CLIP
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
Photoactivatable Ribonucleoside_Enhanced Crosslinking and Immunoprecipitation
PAR-CLIP maps RBP sites on the target RNAs (Hafner et al., 2008). This approach is similar to HITS-CLIP and CLIP-Seq, but it uses much more efficient crosslinking to stabilize the protein-RNA complexes. The requirement to introduce a photoactivatable ribonucleoside limits this approach to cell culture and in vitro systems.
In this method, 4-SU and 6-thioguanosine (6-SG) are incorporated into transcripts of cultured cells. UV irradiation crosslinks 4-SU/6-SG_labeled transcripts to interacting RBPs. The targeted complexes are immunoprecipitated and digested with RNase T1, followed by proteinase K, before RNA extraction. The RNA is reverse-transcribed to cDNA and sequenced. Deep sequencing of cDNA accurately maps RBPs interacting with labeled transcripts.
Advantages:- Highly accurate mapping of RNA-protein interactions
- Labeling with 4-SU/6-SG improves crosslinking efficiency
- Legitimately crosslinked sequences can be identified based on the presence of mutated bases, and mispriming can be filtered bioinformatically (Gillen et al., 2016)
Disadvantages:- Antibodies not specific to the target may precipitate nonspecific complexes
- Limited to cell culture and in vitro systems
- Photoreactive nucleosides can be cytotoxic (Kloetgen et al., 2015)
- High concentrations of 4-SU can inhibit rRNA synthesis and induce a nucleolar stress response (Burger et al., 2013)
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;14:74-89Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236References:Tichon A., Gil N., Lubelsky Y., Havkin Solomon T., Lemze D., et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat Commun. 2016;7:12209Vongrad V., Imig J., Mohammadi P., et al. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages. PLoS One. 2015;10:e0132127Liu N., Dai Q., Zheng G., He C., Parisien M. and Pan T. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015;518:560-564Murakawa Y., Hinz M., Mothes J., et al. RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKKF-kappaB pathway. Nat Commun. 2015;6:7367Kang D., Skalsky R. L. and Cullen B. R. EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival. PLoS Pathog. 2015;11:e1004979Matveeva E., Maiorano J., Zhang Q., et al. Involvement of PARP1 in the regulation of alternative splicing. Cell Discov. 2016;2:15046Porter D. F., Koh Y. Y., VanVeller B., Raines R. T. and Wickens M. Target selection by natural and redesigned PUF proteins. Proc Natl Acad Sci U S A. 2015;112:15868-15873Wang Q., Taliaferro J. M., Klibaite U., Hilgers V., Shaevitz J. W. and Rio D. C. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Proc Natl Acad Sci U S A. 2016;113:5269-5274Lee A. S., Kranzusch P. J. and Cate J. H. eIF3 targets cell-proliferation messenger RNAs for translational activation or repression. Nature. 2015;522:111-114Chu Y., Wang T., Dodd D., Xie Y., Janowski B. A. and Corey D. R. Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells. Nucleic Acids Res. 2015;43:e75Xie H., Lee L., Scicluna P., et al. Novel functions and targets of miR-944 in human cervical cancer cells. Int J Cancer. 2015;136:E230-241Xiong X. P., Vogler G., Kurthkoti K., Samsonova A. and Zhou R. SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function. PLoS Genet. 2015;11:e1005475Chen T., Hao Y. J., Zhang Y., et al. m(6)A RNA Methylation Is Regulated by MicroRNAs and Promotes Reprogramming to Pluripotency. Cell Stem Cell. 2015;16:289-301