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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Knockdown of PTBP3 in human K562 cells

ABSTRACT: PTBP1 is a widely expressed RNA binding protein known to play roles in regulation of alternative pre-mRNA splicing and other post-transcriptional processes. The PTBP2 paralog is expressed in neurons and switching from expression of PTBP1 to PTBP2 regulates a subset of alternative splicing events during neuronal differentiation. The PTBP3 paralog is conserved across vertebrates and is expressed highly in cells of the hematopoietic system. However, the unique non-redundant roles of PTBP3 are currently unclear. We therefore tested the transcriptomic consequences of knocking down PTBP3 in human K562 erythroleukemia cells. K562 cells were treated with two separate siRNAs (Rd5 and Rd9) targeted against all isoforms of PTBP3 or with a control non-specific siRNA (C2). Triplicate samples were used for each of the three experimental conditions. Transcriptome analysis used the Affymetrix HJAY array, allowing detection of changes in alternative splicing and in transcript levels. Despite at least 75% PTBP3 depletion, as monitored by western blot, only modest changes at the transcriptome level were observed.

ORGANISM(S): Homo sapiens

SUBMITTER: Chris Smith

PROVIDER: E-MTAB-4847 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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An Alternative Splicing Event Amplifies Evolutionary Differences Between Vertebrates

Project description:Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here, we show that mammalian-specific skipping of exon 9 of PTBP1 alters its splicing regulatory activities and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon skipping event in an RNA binding regulator directs numerous AS changes between species. The results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems. This study contains two sets of samples: (Set 1) mRNA profiling of human 293 cells subjected to four different conditions in two biological replicates: non-targetting control siRNA, PTBP1 and PTBP2 siRNA, PTBP1 and PTBP2 siRNA with overexpression of full-length human PTBP1, PTBP1 and PTBP2 siRNA with overexpression of exon-excluded human PTBP1. (Set 2) mRNA profiling of chicken DT40 cells with 3 genotypes in two bioligcal replicates: wildtype cells, cells with PTBP1 exon 8 (orthologous to human PTBP1 exon 9) deleted in one allele, and cells with PTBP1 exon 8 deleted in both alleles.

2015-09-01 | E-GEOD-69656 | biostudies-arrayexpress

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Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2 Interact with Distinct Proteins Under Splicing Conditions

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2016-07-03 | E-GEOD-70985 | biostudies-arrayexpress

Splicing analyses of 46C mESCs following PTBP depletion

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2016-07-03 | E-GEOD-71075 | biostudies-arrayexpress

Profiles of gene expression and alternative splicing in K562 cells exposed to 2 mM sodium valproate for 12 hours assessed using exon array

Project description:To investigate the effect of valproate (VPA) treatment on K562 cell line, the growth and survival of the K562 cell line were investigated using the Annexin V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarray. A significant increase in cell apoptosis was observed in valproate exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of observed differentially expressed genes that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed as alternative splicing by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line. Gene expression/alternative splicing in K562 cell line were measured after exposure to 2 mM valproate for 12 hours or left untreated as a control using exon array.

2011-11-11 | E-GEOD-32047 | biostudies-arrayexpress

The MicroRNA miR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-mRNA Splicing

2007-08-04 | GSE8498 | GEO

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq]

Project description:Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.

2013-06-17 | E-GEOD-47997 | biostudies-arrayexpress

Splicing analyses of mESCs, mNPCs, and mMNs

Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. HB9-GFP mESCs were differentiated into mNPCs and mMNs. Poly-A RNA was isolated from isolated populations of mESCs, mNPCs, and mMNs for RNA-sequencing and splicing analyses.

2016-07-03 | E-GEOD-71079 | biostudies-arrayexpress

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