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| Status |
Public on Dec 09, 2011 |
| Title |
In vivo and transcriptome-wide identification of RNA-binding protein target sites [PAR-CLIP] |
| Organism |
Caenorhabditis elegans |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes.
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| Overall design |
Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300µl beads and 150µg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).
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| Contributor(s) |
Anna-Carina J, Marlon S, Desirea M, Dominic G, Guido M, Stefan K, Rajewsky N |
| Citation(s) |
22152485 |
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| Submission date |
Nov 09, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Dominic Gruen |
| E-mail(s) |
dominic.gruen@mdc-berlin.de
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| Organization name |
Max-Delbrueck-Center
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| Street address |
Robert-Roessle-Strasse 10
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| City |
Berlin |
| ZIP/Postal code |
13125 |
| Country |
Germany |
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| Platforms (1) |
| GPL9269 |
Illumina Genome Analyzer II (Caenorhabditis elegans) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE33543 |
In vivo and transcriptome-wide identification of RNA-binding protein target sites |
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| Relations |
| SRA |
SRP009256 |
| BioProject |
PRJNA154339 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE33569_RAW.tar |
384.2 Mb |
(http)(custom) |
TAR (of FA) |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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