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

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.

ORGANISM(S): Mus musculus

PROVIDER: GSE70022 | GEO | 2016/03/15

SECONDARY ACCESSION(S): PRJNA287505

REPOSITORIES: GEO

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

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. 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.

2016-03-15 | E-GEOD-76222 | biostudies-arrayexpress

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

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

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [RBM47 KD]

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Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [RBM47 KD]

Project description:RBM47 is a RNA binding protein that is known to regulate alternative splicing. To study the genome wide regulatory role of RBM47 in alternative splicing and determine the potential function of RBM47 during EMT, we used lentiviral shRNAs to knockdown RBM47 in H358 cells and performed RNA-seq in biological triplicates. We used lentiviral based shRNAs to knockdown RBM47 in human H358 cells. We harvested total RNA and protein from RBM47 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-75491 | biostudies-arrayexpress

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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]

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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]

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Co-regulation of alternative splicing by hnRNPM and ESRP1 during EMT

Project description:The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome-scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they co-regulate a set of cassette exon events, with the majority showing discordant splicing regulation. hnRNPM discordantly regulated splicing events show a positive correlation with splicing during EMT while concordant splicing events do not, highlighting the antagonistic role of hnRNPM and ESRP1 during EMT. Motif enrichment analysis near co-regulated exons identifies guanine-uridine rich motifs downstream of hnRNPM-repressed and ESRP1-enhanced exons, supporting a model of competitive binding to these cis-elements to antagonize alternative splicing. The set of co-regulated exons are enriched in genes associated with cell-migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of co-regulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtypes. In conclusion, hnRNPM and ESRP1 co-regulate antagonistically a set of alternative splicing events that occur during EMT. This regulation is likely mediated through competition for the same intronic binding sites downstream of variable exons. hnRNPM and ESRP1 regulated splicing events are associated with breast cancer survival.

2018-08-01 | GSE112516 | GEO

Transcriptomic Analysis of PNN- and ESRP1-regulation in Human Corneal Epithelial Cells

Project description:Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In human corneal epithelial cells (HCET), ESRP1 and PNN displayed close localization in and around nuclear speckles and their physical association in protein complexes was identified. In this study, gene expression profiling was performed to identify PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells. Immortalized human corneal epithelial cells harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript level and splicing pattern of specific subsets of genes with significant overlaps in their candidate targets. Our data suggest that ESRP1 and PNN modulate alternative splicing of a specific subset of exons, but not general splicing events. ESRP1 and PNN may together participate in the regulation of epithelial-specific splicing program in a genome-wide fashion. Parental HCET, shRNA-PNN HCET, and shRNA-ESRP1 HCET cells were cultured for 3 days with/without doxycycline. Total RNA was isolated from four biological replicates of each sample group and then subjected to hGlue3_0 transcriptome array analysis.

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