Transcriptome-wide mapping of hnRNP C and U2AF65 using iCLIP
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR196173.fastq.gz | Fastqsanger.gz | |||
| ERR196174.fastq.gz | Fastqsanger.gz | |||
| ERR196175.fastq.gz | Fastqsanger.gz | |||
| ERR196176.fastq.gz | Fastqsanger.gz | |||
| ERR196177.fastq.gz | Fastqsanger.gz |
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Project description:iCLIP experiment to assess the binding of the highly abundant nuclear RNA-binding protein hnRNP C and core splicing factor U2AF65 on a genomic scale. To investigate how both proteins compete for binding at a subset of sites, U2AF65 iCLIP experiments were performed from both HNRNPC knockdown and control HeLa cells.
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Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene, we performed hnRNP H iCLIP with RON wildtype minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two replicates of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina HiSeq2000 (75-nt single-end reads).
2018-06-27 | E-MTAB-6220 | biostudies-arrayexpress
Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene with either wildtype or hnRNP H binding site mutant background, we performed hnRNP H iCLIP with RON wildtype or mutant minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two (G331C and G348C) or three replicates (wildtype and G305A) of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina MiSeq or NextSeq500 with 75-nt single-end reads.
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Project description:Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF65 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We established 'in vitro iCLIP' experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF65 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We quantitatively measure U2AF65 affinities at hundreds of binding sites, and compare in vitro and in vivo binding landscapes by mathematical modelling. We find that trans-acting RBPs extensively regulate U2AF65 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of intronic regions. Using machine learning, we identify novel trans-acting RBPs (including FUBP1, BRUNOL6 and PCBP1) that modulate U2AF65 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.
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Project description:Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF65 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We established 'in vitro iCLIP' experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF65 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We quantitatively measure U2AF65 affinities at hundreds of binding sites, and compare in vitro and in vivo binding landscapes by mathematical modelling. We find that trans-acting RBPs extensively regulate U2AF65 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of intronic regions. Using machine learning, we identify novel trans-acting RBPs (including FUBP1, BRUNOL6 and PCBP1) that modulate U2AF65 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.
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