Dataset Information

Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

ABSTRACT: Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.

SUBMITTER: Garzia A

PROVIDER: S-EPMC5393971 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

Garzia Aitor A Meyer Cindy C Morozov Pavel P Sajek Marcin M Tuschl Thomas T

Methods (San Diego, Calif.) 20161017

Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data ...[more]

PMID: 27765618

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Project description: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. 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 22ÂºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ÂºC 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), 300Âµl beads and 150Âµg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ÂºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).

Dataset Information

Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

Publications

Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

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