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Gene Expression and Exon Splicing Change Analysis of Mouse N2A Cell Transcriptome upon Polypyrimidine tract-binding protein depletion.

ABSTRACT: Purpose : Splicing factors regulate splice site choices in pre-mRNA and determine final exon set in mRNA. To understand mechanisms of splicing regulation, it is important to identify and characterize exon targets of splicing factors. Recently, development of RNA-seq technology enables researchers to investigate exon splicing profiles as well as gene expression profiles in transcriptome-wide. The goal of this study is to investigate transcriptome changes by splicing factors, Polypyrimidine Tract Binding proteins (PTB). In this study, we analyzed exon and gene expression changes followed by Ptbp1 knock down. Methods : The knockdown experiment was performed in mouse neuroblastoma (N2A) cells. Total RNA was collected from cells and further treated with DNase I to avoid DNA contamination. RNA-seq libraries were constructed in a strand specific way using dUTP and Uracil-Specific Excision Reagent enzyme. The libraries were subjected to 100bp paired-end sequencing (Illumina HiSeq2000 platform). Poly(A)-mRNA and exon profiles of N2A mouse blastoma cells in two samples: shRNA transfection control, single knock down of ptbp1. RNA-seq libraries were generated in strand specific way using dUTP and USER enzyme and sequenced using Illumina HiSeq2000.

ORGANISM(S): Mus musculus

SUBMITTER: Areum Han

PROVIDER: E-GEOD-45119 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene Expression and Exon Splicing Change Analysis of Mouse N2A Cell Transcriptome upon Polypyrimidine tract-binding protein depletion (II)

Project description:Purpose: Splicing factors regulate splice site choices in pre-mRNA and determine final exon set in mRNA. To understand mechanisms of splicing regulation, it is important to identify and characterize exon targets of splicing factors. Recently, development of RNA-seq technology enables researchers to investigate exon splicing profiles as well as gene expression profiles in transcriptome-wide. The goal of this study is to investigate transcriptome changes by splicing factors, Polypyrimidine Tract Binding proteins (PTB). In this study, we analyzed exon and gene expression changes followed by Ptbp1, Ptbp2, and both Ptbp1 & Ptbp2 knock down. Methods: The knockdown experiment was performed in mouse neuroblastoma (N2A) cells. Total RNA was collected from cells and further treated with DNase I to avoid DNA contamination. RNA-seq libraries were constructed in a strand specific way using dUTP and Uracil-Specific Excision Reagent enzyme. The libraries were subjected to 100bp paired-end sequencing (Illumina HiSeq2000 platform).

2014-12-02 | GSE52856 | GEO

Gene Expression and Exon Splicing Change Analysis of Mouse N2A Cell Transcriptome upon Polypyrimidine tract-binding protein depletion.

Project description:Purpose : Splicing factors regulate splice site choices in pre-mRNA and determine final exon set in mRNA. To understand mechanisms of splicing regulation, it is important to identify and characterize exon targets of splicing factors. Recently, development of RNA-seq technology enables researchers to investigate exon splicing profiles as well as gene expression profiles in transcriptome-wide. The goal of this study is to investigate transcriptome changes by splicing factors, Polypyrimidine Tract Binding proteins (PTB). In this study, we analyzed exon and gene expression changes followed by Ptbp1 knock down. Methods : The knockdown experiment was performed in mouse neuroblastoma (N2A) cells. Total RNA was collected from cells and further treated with DNase I to avoid DNA contamination. RNA-seq libraries were constructed in a strand specific way using dUTP and Uracil-Specific Excision Reagent enzyme. The libraries were subjected to 100bp paired-end sequencing (Illumina HiSeq2000 platform).

2013-12-01 | GSE45119 | GEO

Gene expression analyses of HB9-GFP D2 mMN cultures following Pbx1 exon 7 inclusion

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 mESC were transiently transfected with Cas9 and guide RNA sequences to generate heterozygous deletions at the Pbx1 intron 6 locus (Pbx1 I6 +/-). Wild type and (n=3) or Pbx1 I6 +/- (n=5) clones were differentiated in MN media. Samples were harvested at Day 2 of differentiation, and poly-A RNA was isolated for RNA-sequencing and gene expression analyses.

2016-07-03 | E-GEOD-70883 | biostudies-arrayexpress

A global regulatory mechanism for activating an exon network required for neurogenesis

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An Alternative Splicing Event Amplifies Evolutionary Differences Between Vertebrates

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

Ptpb1 iCLIP in 46C mESCs and mNPCs

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2016-07-03 | E-GEOD-71178 | biostudies-arrayexpress

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Project description:Changes in alternative splicing in breast cancer cells expressing control, empty vector or Flag-tagged wild type RBM47 were analyzed using paired-end, 100bp RNAseq. Related data published together with these data are found in GSE53779 Triplicate RNAseq libraries were prepared from non-clonal brain metastatic breast cancer cells stably expressing empty-vector, and a clonal cell line (wt#10) expressing Flag-tagged, wild-type RBM47 under a doxycline-inducible promoter, both treated for three days with doxycycline to induce transgene expression

2014-06-11 | E-GEOD-58379 | biostudies-arrayexpress

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

Project description:RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTBP proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. How the two related proteins regulate different sets of neuronal exons is unclear. The distinct splicing activities of PTBP1 and PTBP2 can be recapitulated in an in vitro splicing system with the differentially regulated N1 exon of the c-src pre-mRNA. Here, we conducted experiments under these in vitro splicing conditions to identify PTBP1 and PTBP2 interacting partner proteins.

2022-01-20 | PXD029899 | Pride

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.

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

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