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

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE38355 | GEO | 2012/06/01

SECONDARY ACCESSION(S): PRJNA167768

REPOSITORIES: GEO

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

Project description: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. We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced

2012-06-01 | E-GEOD-38355 | biostudies-arrayexpress

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

2012-06-01 | GSE38356 | GEO

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

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

Project description: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. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with HEK293 cell lines stably expressing HIS/FLAG/HA-tagged ALKBH5, C17orf85, C22orf28, CAPRIN1, and ZC3H7B. We used 4-thiouridine (4SU) to enhance the crosslink and generated two PAR-CLIP cDNA libraries per protein.

2012-06-01 | E-GEOD-38201 | biostudies-arrayexpress

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

Project description: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).

2012-06-01 | E-GEOD-38356 | biostudies-arrayexpress

Differential Protein Occupancy Profiling of the mRNA Transcriptome

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Differential Protein Occupancy Profiling of the mRNA Transcriptome

Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We have 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 (Baltz and Munschauer et al. 2012). Our current work focuses on streamlining and extending protein occupancy profiling on poly(A)-RNA. Our objectives are to identify previously unknown protein-bound transcripts and, more importantly, to assess global and local differences in protein occupancy across different biological conditions. To this end, we have implemented poppi, the first pipeline for differential analysis of protein occupancy profiles. We have applied our analysis pipeline to pinpoint changes in occupancy profiles of MCF7 cells against already published HEK293 cells. We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled MCF7 cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced.

2013-12-05 | E-GEOD-49831 | biostudies-arrayexpress

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

Project description:This SuperSeries is composed of the following subset Series: GSE38356: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [RNA-seq] GSE38201: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [PAR-CLIP] GSE38355: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [protein occupancy profiling] Refer to individual Series

2012-05-31 | E-GEOD-38157 | biostudies-arrayexpress

Escherichia coli Tiling Array for In vivo Protein Occupancy Display

Project description:Using a high-density tiling array containing 25-mer oligonucleotides at the resolution of one every four base pairs across the entire genome, we developed In vivo Protein Occupancy Display (IPOD), a technology that reveals protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites. Application to Escherichia coli reveals thousands of protein occupancy peaks, highly enriched within and in close proximity to non-coding regulatory regions.

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Protein occupancy of Mycoplasma pneumoniae chromosome

Project description:Protein occupancy is a means of identifying all the regions in the genome that are bound to proteins. A simple protocol is used whereby cross-linked chromatin is digested with DNase to remove all non-crosslinked, naked, DNA. To distinguish the regions that are bound by RNA polymerase from the rest, a ChAPseq (chromatin affinity precipitation) with TAP-tagged alpha subunit of RNApol, was performed.

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