Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [RNA-seq]

ABSTRACT: Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. To obtain a more detailed picture of the RNA present in the pooled precipitates of four consecutive oligo(dT)-purifications, we constructed a cDNA library by random priming of 4-thiouridine (4SU)- and 6-thioguanosine (6SG)-labeled RNA derived from UV-irradiated (365 nm)and non-irradiated cells. Digital gene expression analysis of the cDNA library of non-irradiated cells, labeled with 4SU and 6SG, was performed. To monitor the incorporation of photoreactive nucleotides into mRNA, we isolated 4SU- and 6SG-labeled RNA from the oligo(dT) precipitate of non-crosslinked cells by biotinylation and streptavidin purification (Dolken et al., 2008).

ORGANISM(S): Homo sapiens

SUBMITTER: Markus Landthaler

PROVIDER: E-GEOD-38356 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.

Baltz Alexander G AG Munschauer Mathias M Schwanhäusser Björn B Vasile Alexandra A Murakawa Yasuhiro Y Schueler Markus M Youngs Noah N Penfold-Brown Duncan D Drew Kevin K Milek Miha M Wyler Emanuel E Bonneau Richard R Selbach Matthias M Dieterich Christoph C Landthaler Markus M

Molecular cell 20120601 5

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearl ...[more]

PMID: 22681889

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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

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [RNA-seq]

Publications

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.

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