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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [ESRP DKO]

ABSTRACT: Alternative splicing (AS) plays a critical role in cell fate transitions, development and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS during reprogramming, including a splicing program that is unique to transgene-independent iPS cells. Changes in the expression of alternative splicing factors Zcchc24, Esrp1, Mbnl1/2 and Rbm47 were demonstrated to be key contributors to phase-specific AS. RNA binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1.These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency. ES cells from 3 independent E3.5 blastocysts from either Control (Esrp1 WT/WT; Esrp2 -/-) or Esrp DKO (Esrp1 floxed/floxed; Esrp2 -/-) were transfected with pLVX-EGFP-Cre, puro selected and RNA was isolated 6 days later.

ORGANISM(S): Mus musculus

SUBMITTER: Yi Xing

PROVIDER: E-GEOD-76222 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [iPS]

Project description:Alternative splicing (AS) plays a critical role in cell fate transitions, development and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS during reprogramming, including a splicing program that is unique to transgene-independent iPS cells. Changes in the expression of alternative splicing factors Zcchc24, Esrp1, Mbnl1/2 and Rbm47 were demonstrated to be key contributors to phase-specific AS. RNA binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1.These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency. Mouse embryo fibroblasts were isolated from an E12.5 embryo that was homozygous for a TET-OP-OKSM polycistronic transgene targeted to the collagen 1A locus and homozygous for rtTA targeted to the Rosa26 locus. Therefore the expression of the â??4 factors/Yamanaka factorsâ?? that induce pluripotency can be induced with doxycycline treatment. Using this model we isolated RNA in triplicate over a timecourse of 0, 4, 7, 10, 15, 20, days of dox treatment as well as 3 transgene independent clones. Ssea1 MACS purification (Miltenyi Biotec) was used at each time point except zero.

2016-03-15 | E-GEOD-70022 | biostudies-arrayexpress

Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [iPS]

2016-03-15 | GSE70022 | GEO

Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [ESRP DKO]

2016-03-15 | GSE76222 | GEO

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [ESRP KD]

Project description:Epithelial specific splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) are important regulators of alternative splicing during EMT. To study the alternative splicing events regulated by ESRP1/2 at a genome wide scale, we used lentiviral shRNAs to knockdown ESRP1/2 in H358 cells and performed RNA-seq in biological triplicates. We used lentiviral based shRNAs targeting ESRP1 and ESRP2 to knockdown both regulators in human H358 cells. We harvested total RNA and protein from ESRP1/2 knockdown and control knockdown in biological triplicates. We made cDNA libraries for each replicate and subjected them to RNA-seq.

2016-04-05 | E-GEOD-75489 | biostudies-arrayexpress

Knockout mice reveal an essential role for Epithelial splicing regulatory proteins (Esrps) in mammalian development and epithelial splicing in vivo

Project description:Tissue- and cell-type specific regulators of alternative splicing (AS) are an essential layer of posttranscriptional gene regulation necessary for normal cellular function, patterning, and development. Here we report the Epithelial splicing regulatory proteins (Esrps) are required for patterning of multiple organs, with loss of both paralogs, Esrp1 and Esrp2, resulting in increasingly severe phenotypes. Global profiling of the Esrp splicing regulatory network from total epidermis revealed varied splicing sensitivity of Esrp targets upon loss of Esrp1 or double knockout. This may explain the progressive phenotypes seen in Esrp knockout mice, and these mice provide a unique genetic tool to evaluate functional consequences of epithelial splicing events in vivo. RNA from purified total epidermis (basal keratinocyte layer to cornified layer) of E18.5 mouse embryos were harvested by Trizol extraction. (Esrp1+/+, Esrp2-/- (n=2), Esrp1-/-, Esrp2+/+ (n=3), Esrp1-/-, Esrp2+/- (n=2), and Esrp1-/-, Esrp2-/- (n=2). 1 ug of total RNA was used for for RNA-seq library preparation using the TruSeq™ Stranded mRNA LT Sample Prep Kit (Illumina). 100x2 bp paired-end RNA-seq reads were generated on a HiSeq 2000 sequencer.

2015-09-08 | E-GEOD-64357 | biostudies-arrayexpress

Ablation of the epithelial-specific splicing factor Esrp1 results in ureteric branching defects and reduced nephron number

Project description:Evaluation of splicing in ureteric epithelium in Esrp1;Esrp2 double knockout mice compared to Esrp1+/+;Esrp2-/- control mice.

2016-05-21 | GSE81716 | GEO

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [ESRP KD]

2016-04-05 | GSE75489 | GEO

The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate alternative splicing events

Project description:Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are recently discovered epithelial-specific RNA-binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, CD44, and CTNND1 transcripts. To catalogue a larger set of splicing events under the regulation of the ESRPs, we profiled splicing changes induced by RNA interference-mediated knockdown of ESRP1 and ESRP2 expression in a human epithelial cell line using the splicing-sensitive Affymetrix Exon ST1.0 Arrays. Analysis of the microarray data using the previously described MADS tool resulted in the identification of over a hundred candidate ESRP-regulated splicing events. We were able to independently validate 37 of these targets by RT-PCR. The ESRP-regulated events encompass all known types of alternative splicing events. Importantly, a number of these regulated splicing events occur in gene transcripts that encode proteins with well-described roles in the regulation of actin cytoskeleton organization, cell-cell adhesion, cell polarity, and cell migration. In sum, this work reveals a novel list of transcripts differentially spliced in epithelial and mesenchymal cells, implying that coordinated alternative splicing plays a critical role in determination of cell type identity. Keywords: control / knockdown comparison Short interfering knockdown of ESRP1 and ESRP2 in human PNT2 prostatic epithelium cells was performed as described before (Warzecha et al., 2009, Molecular Cell 33:591-601). The efficiency of ESRP1 and ESRP2 knockdown was monitored by quantitative RT-PCR as described before (Warzecha et al., 2009, Molecular Cell 33:591-601). In all cases the efficiency of the knockdown was close to 80%. We conducted Exon array profiling on RNAs from four siESRP1/2-treated samples and four siGFP controls.

2010-08-18 | E-GEOD-17468 | biostudies-arrayexpress

Roles of ESRPs in maintenance of epidermal transcriptome

Project description:The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3’ and 5’ untranslated regions (UTRs) of genes involved in epithelial cell function, suggesting that its post-transcriptional functions extend beyond splicing regulation.

2022-09-12 | GSE212306 | GEO

Identification of ESRP1 targets in the epidermis

2022-09-12 | GSE212304 | GEO

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