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. 2016;1358:153-73.
doi: 10.1007/978-1-4939-3067-8_10.

PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites

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Free PMC article

PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites

Charles Danan et al. Methods Mol Biol. .
Free PMC article

Abstract

During post-transcriptional gene regulation (PTGR), RNA binding proteins (RBPs) interact with all classes of RNA to control RNA maturation, stability, transport, and translation. Here, we describe Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a transcriptome-scale method for identifying RBP binding sites on target RNAs with nucleotide-level resolution. This method is readily applicable to any protein directly contacting RNA, including RBPs that are predicted to bind in a sequence- or structure-dependent manner at discrete RNA recognition elements (RREs), and those that are thought to bind transiently, such as RNA polymerases or helicases.

Keywords: Binding site; Cross-linking and immunoprecipitation (CLIP); Noncoding RNA; Photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP); Posttranscriptional gene regulation (PTGR); RNA; RNA recognition element (RRE); RNA-binding protein (RBP); mRNA.

Figures

Fig. 1
Fig. 1
Outline of the PAR-CLIP methodology. PAR-CLIP begins with incorporation of photoactivatable thioribonucleosides into nascent transcripts followed by cross-linking with long-wavelength >310 nm UV. Cross-linked RNA–RBP complexes are isolated by immunoprecipitation and further purified by SDS-PAGE. After recovery from the purified radioactive band, the RNA is carried through a small RNA cDNA library preparation protocol for sequencing. Reverse transcription of cross-linked RNA with incorporated photoactivatable thioribonucleosides, followed by PCR amplification, leads to a characteristic mutation (T-to-C when using 4SU and G-to-A when using 6SG) that is used to identify the RNA recognition elements

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