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Transcriptome-wide mapping of RNA binding sites of the RNA-binding protein Unkempt using iCLIP-seq

ABSTRACT: Transcriptome-wide mapping of RNA binding sites of the RNA-binding protein Unkempt using iCLIP-seq

PROVIDER: PRJEB5286 | ENA |

REPOSITORIES: ENA

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Transcriptome-wide mapping of RNA binding sites of the RNA-binding protein Unkempt using iCLIP-seq

Project description:iCLIP experiments tomap the RNA binding sites of the RNA-binding protein Unkempt across the transcriptome in SH-SY5Y cells, HeLa cells with ectopic Unk expression and mouse E15 embryonic brain samples. Expression of Unk is normally largely restricted to the nervous system. We therefore mapped the binding sites in human SH-SY5Y and mouse E15 brain to detect its physiological binding sites (in SH-SY5Y, we also performed the RNAseq experiment upon Unk knockdown). HeLa cells on the other hand normally don't express Unk, but convert to neuron-like shape when the protein is ectopically expressed. So, here we hoped to identify those binding events (and hence target transcripts) that are critical for this morphological transformation.

2015-03-09 | E-MTAB-2279 | biostudies-arrayexpress

iCLIP transcriptome-wide mapping of UNR (CSDE1) binding sites

Project description:iCLIP transcriptome-wide mapping of UNR (CSDE1) binding sites

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iCLIP transcriptome-wide mapping of UNR (CSDE1) binding sites

Project description:Analysis of UNR (CSDE1) binding to RNA targets by iCLIP transcriptome-wide mapping.

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Transcriptome-wide mapping of hnRNP C and U2AF65 using iCLIP

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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Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

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

High-resolution analysis of SRSF3 and SRSF4 binding sites by iCLIP

Project description:Endogenous SRSF3 and SRSF4 RNA binding sites were identified genome-wide by iCLIP in P19 BAC trangenic cell lines using GFP-epitope tag.

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Mapping hnRNP H binding sites on RON wildtype minigene and RON mutant minigenes via iCLIP.

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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Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

Project description:Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

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Project description:iCLIP analysis of RNA binding sites of Stau2 protein in mouse brain cells

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Mapping the targeting transcripts and binding sites of ALKBH5 by individual-nucleotide resolution UV crosslinking and immunoprecipitation-based sequencing (iCLIP-seq)

Project description:To investigate whether the ALKBH5-mediated metabolic changes via demethylation of its target mRNA(s) is important in regulating viral response, we mapped the targeting transcripts and binding sites of ALKBH5 in the virus infected cells and uninfected cells by using iCLIP-seq. Biological replicates of iCLIP-seq confirmed that OGDH is the direct targeting transcript of ALKBH5 and the binding capacity of ALKBH5 to OGDH mRNA was decreased upon viral infection. By combining the ALKBH5-iCLIP-seq results with m6A-seq data, we identified that there are overlapped ALKBH5-iCLIP peaks and m6A-seq peaks on OGDH mRNA. Furthermore, other mRNAs, such as GOT2 mRNA, were also the ALKBH5’s targeting transcripts and m6A demethylation substrates. GOT2 is an important metabolic enzyme well known to be involved in host response to viral infection. These data demonstrated that ALKBH5-mediated metabolic rewiring via demethylation of target OGDH mRNA and other transcripts is important in regulating cellular viral replication.

2019-08-22 | GSE134754 | GEO

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