PAR-CLIP

Par-CLIP

PAR-CLIP maps RBP sites on the target RNAs. 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.

Pros:

• 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 bioinformatically¹

Cons:

• Antibodies not specific to the target may precipitate nonspecific complexes
• Limited to cell culture and in vitro systems
• Photoreactive nucleosides can be cytotoxic²
• High concentrations of 4-SU can inhibit rRNA synthesis and induce a nucleolar stress response³

• Par-CLIP: Hafner M., Landgraf P., Ludwig J., Rice A., Ojo T., et al. (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44: 3-12
• 1. 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
• 2. Kloetgen A., Munch P. C., Borkhardt A., Hoell J. I. and McHardy A. C. Biochemical and bioinformatic methods for elucidating the role of RNA-protein interactions in posttranscriptional regulation. Brief Funct Genomics. 2015;14:102-114
• 3. Burger K., Muhl B., Kellner M., et al. 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. RNA Biol. 2013;10:1623-1630

• TruSeq RNA Library Prep Kit
• TruSeq Small RNA Sample Prep Kit
• TruSeq Stranded RNA
• TruSeq DNA PCR-Free library prep kit
• TruSeq Nano DNA Library Prep kit

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2. 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

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5. 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
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11. 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
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