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CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of PTB / hnRNP I

ABSTRACT: CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of PTB / hnRNP I

PROVIDER: PRJEB7865 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR689063.fastq.gz	Fastqsanger.gz
	ERR689064.fastq.gz	Fastqsanger.gz
	ERR689065.fastq.gz	Fastqsanger.gz
	ERR689066.fastq.gz	Fastqsanger.gz
	ERR689067.fastq.gz	Fastqsanger.gz

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CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of PTB / hnRNP I

Project description:PTB iCLIP-seq in HeLa cells

2014-12-01 | E-MTAB-3108 | biostudies-arrayexpress

PTB CLIP-seq

Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells. Examination of PTB-RNA binding in Hela cells using CLIP-seq (Cross-Linking ImmunoPrecipitation coupled with high-throughput sequencing) method. Peaks: The four alignment files (linked as supplementary files on Sample records) were combined together for peak finding, as we found that most of the monomeric and dimeric tags are similarly distributed in the genome with high pearson correlation coefficient. The method to detect the peaks above gene-specific randomized background was similar to (Yeo et al., 2009) and described in the paper (Xue et al., 2009).

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Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37°C. For iCLIP, HeLa cells expressing eIF4A3-GFP or PTB-GFP were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries for eIF4A3 and PTB were sequenced with 50 bp on an Illumina HiSeq instrument.

2015-08-11 | E-MTAB-2599 | biostudies-arrayexpress

Individual-nucleotide resolution CLIP and high-throughput sequencing to map hnRNP L-RNA interactions in HeLa cells

Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells

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iCLIP of eIF4A3 and PTB in HeLa cells

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Project description:Transcription profiling of human HeLa cells (cervical cancer cell line) transfected with a plasmid expressing shRNAs cloned into the pSuper expression vector compared to emprty vector negative controls for transfection. Four different RNA interference treatments targetted: A1 hnRNP (HNRNPA1, Heterogeneous nuclear ribonucleoprotein A1); FUS (fusion gene, involved in t(12;16) in malignant liposarcoma); H hnRNP (HNRNPH1); and p68 helicase (DDX5, DEAD (Asp-Glu-Ala-Asp) box polypeptide 5). Keywords: genetic modification

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Project description:CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of Matrin3

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Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated microRNA Circuits

Project description:The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. Examination of PTB regulated AGO2/microRNA targeting in Hela cells by CLIP-seq (two biological replicates) , paired-end RNA-seq (control and PTB knockdown) and 3’end stability RNA-seq (control and PTB knockdown)

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