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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]

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

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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.

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

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