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In JoVE (1)
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Articles by Greg S. Wardle in JoVE
PAR-Clip - En metod för att identifiera transkriptom hela bindningar på RNA-proteiner
Markus Hafner1, Markus Landthaler2, Lukas Burger3, Mohsen Khorshid3, Jean Hausser4, Philipp Berninger4, Andrea Rothballer1, Manuel Ascano1, Anna-Carina Jungkamp2, Mathias Munschauer2, Alexander Ulrich1, Greg S. Wardle1, Scott Dewell5, Mihaela Zavolan3, Thomas Tuschl1
1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University
RNA-transkript är föremål för omfattande posttranscriptional reglering som förmedlas av en mängd gränsöverskridande agerar RNA-bindande proteiner (RBPs). Här presenterar vi en generaliserbar metod för att identifiera exakt och på ett transkriptom-omfattande RNA-bindning med RBPs.
Other articles by Greg S. Wardle on PubMed
Nature. Oct, 2009 | Pubmed ID: 19812667
The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.
Cell. Apr, 2010 | Pubmed ID: 20371350
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.