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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.

Cell | 2010

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.

Pubmed ID: 20371350 RIS Download

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Associated grants

  • Agency: NIGMS NIH HHS, United States
    Id: P01 GM073047-01
  • Agency: NIMH NIH HHS, United States
    Id: R01 MH080442
  • Agency: NIMH NIH HHS, United States
    Id: MH08442
  • Agency: NIGMS NIH HHS, United States
    Id: GM073047
  • Agency: NIGMS NIH HHS, United States
    Id: P01 GM073047
  • Agency: NIMH NIH HHS, United States
    Id: R01 MH080442-01
  • Agency: Howard Hughes Medical Institute, United States

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Addgene (tool)

RRID:SCR_002037

Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

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TargetScan (tool)

RRID:SCR_010845

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