Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP

• Published in: Cell Vol. 141; no. 1; pp. 129 - 141
• Main Authors: Hafner, Markus, Landthaler, Markus, Burger, Lukas, Khorshid, Mohsen, Hausser, Jean, Berninger, Philipp, Rothballer, Andrea, Ascano, Manuel, Jungkamp, Anna-Carina, Munschauer, Mathias, Ulrich, Alexander, Wardle, Greg S., Dewell, Scott, Zavolan, Mihaela, Tuschl, Thomas
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.04.2010
• Subjects: Base Sequence; Cross-Linking Reagents - metabolism; Genetic Techniques; Humans; MicroRNAs - metabolism; Molecular Sequence Data; Nucleosides - metabolism; Point Mutation; PROTEINS; Regulatory Sequences, Ribonucleic Acid; RNA; RNA, Untranslated - genetics; RNA-Binding Proteins - metabolism; Sequence Alignment; SYSBIO; RNA; SYSBIO; PROTEINS
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2010.03.009

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases. [Display omitted] ► PAR-CLIP is a transcriptome-wide crosslinking method for RNA-binding proteins (RBP) ► It is based on incorporation of photoactivatable nucleoside analogs into nascent RNA ► Characteristic sequence transitions in the prepared cDNA reveal the precise binding site ► We deduced binding motifs and preferences for 5 different RBP families