Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.
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.
Hafner
Markus
M
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA.
Landthaler
Markus
M
Burger
Lukas
L
Khorshid
Mohsen
M
Hausser
Jean
J
Berninger
Philipp
P
Rothballer
Andrea
A
Ascano
Manuel
M
Jungkamp
Anna-Carina
AC
Munschauer
Mathias
M
Ulrich
Alexander
A
Wardle
Greg S
GS
Dewell
Scott
S
Zavolan
Mihaela
M
Tuschl
Thomas
T
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000378:metabolism; D017354:Point Mutation; D022661:RNA, Untranslated / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D016415:Sequence Alignment
D003432:Cross-Linking Reagents; D035683:MicroRNAs; D009705:Nucleosides; C490832:QKI protein, human; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid