» Articles » PMID: 24581442

PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-by-step Protocol to the Transcriptome-wide Identification of Binding Sites of RNA-binding Proteins

Overview
Journal Methods Enzymol
Specialty Biochemistry
Date 2014 Mar 4
PMID 24581442
Citations 55
Authors
Affiliations
Soon will be listed here.
Abstract

We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010). Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.

Citing Articles

Profiling the epigenome using long-read sequencing.

Liu T, Conesa A Nat Genet. 2025; 57(1):27-41.

PMID: 39779955 DOI: 10.1038/s41588-024-02038-5.


Molecular insights into regulatory RNAs in the cellular machinery.

Yang S, Kim S, Yang E, Kang M, Joo J Exp Mol Med. 2024; 56(6):1235-1249.

PMID: 38871819 PMC: 11263585. DOI: 10.1038/s12276-024-01239-6.


Reversible oxidative dimerization of 4-thiouridines in tRNA isolates.

Bessler L, Gross J, Kampf C, Opatz T, Helm M RSC Chem Biol. 2024; 5(3):216-224.

PMID: 38456039 PMC: 10915967. DOI: 10.1039/d3cb00221g.


The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes.

Yeter-Alat H, Belgareh-Touze N, Huvelle E, Banroques J, Tanner N Genes (Basel). 2023; 14(8).

PMID: 37628617 PMC: 10454743. DOI: 10.3390/genes14081566.


MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B.

Masalha M, Meningher T, Mizrahi A, Barzilai A, Tabibian-Keissar H, Gur-Wahnon D Int J Mol Sci. 2022; 23(23).

PMID: 36499729 PMC: 9741271. DOI: 10.3390/ijms232315401.


References
1.
Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C . Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods. 2007; 44(1):3-12. PMC: 2847350. DOI: 10.1016/j.ymeth.2007.09.009. View

2.
Moore M . From birth to death: the complex lives of eukaryotic mRNAs. Science. 2005; 309(5740):1514-8. DOI: 10.1126/science.1111443. View

3.
Ule J, Jensen K, Ruggiu M, Mele A, Ule A, Darnell R . CLIP identifies Nova-regulated RNA networks in the brain. Science. 2003; 302(5648):1212-5. DOI: 10.1126/science.1090095. View

4.
Favre A, Moreno G, Blondel M, Kliber J, Vinzens F, Salet C . 4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells. Biochem Biophys Res Commun. 1986; 141(2):847-54. DOI: 10.1016/s0006-291x(86)80250-9. View

5.
Landthaler M, Gaidatzis D, Rothballer A, Chen P, Soll S, Dinic L . Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. 2008; 14(12):2580-96. PMC: 2590962. DOI: 10.1261/rna.1351608. View