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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).

ORGANISM(S): Mus musculus

PROVIDER: GSE45119 | GEO | 2013/12/01

SECONDARY ACCESSION(S): PRJNA192958

REPOSITORIES: GEO

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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). 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.

2013-12-01 | E-GEOD-45119 | biostudies-arrayexpress

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

PTB CLIP-seq

2009-12-24 | GSE19323 | GEO

PTB CLIP-seq

Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells. Examination of PTB-RNA binding in Hela cells using CLIP-seq (Cross-Linking ImmunoPrecipitation coupled with high-throughput sequencing) method. Peaks: The four alignment files (linked as supplementary files on Sample records) were combined together for peak finding, as we found that most of the monomeric and dimeric tags are similarly distributed in the genome with high pearson correlation coefficient. The method to detect the peaks above gene-specific randomized background was similar to (Yeo et al., 2009) and described in the paper (Xue et al., 2009).

2009-12-24 | E-GEOD-19323 | biostudies-arrayexpress

MADS: a New and Improved Method for Analysis of Differential Alternative Splicing by Exon-tiling Microarrays

Project description:We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB. From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing. Keywords: control / knockdown comparison Short hairpin knockdown of PTB in mouse N2A neuroblastoma cells was performed as described before (Boutz et al., 2007, Genes Dev 21:1636-1652). The efficiency of the PTB knockdown was monitored by western blot using PTB-NT primary antibody and Cy5 labeled secondary antibody (GE Life Sciences). The blots were imaged using Typhoon 9410 (GE Life Sciences). The band intensities were measured using ImageQuant and normalized to GAPDH. In all cases the efficiency of the knockdown was close to 80% (data not shown). We conducted Exon array profiling on RNAs from three shRNA-PTB treated samples and three mock-treated controls (using empty vectors).

2008-05-09 | E-GEOD-11344 | biostudies-arrayexpress

Exon array analysis of N2A cells after knocking down FUS

Project description:To analyze the role of Fus, Ewsr1, and Taf15 in alternative RNA processing, we performed exon array analysis in N2A cells using exon arrays. N2A cells were transfected with siRNA using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions.

2020-08-10 | GSE60694 | GEO

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

Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

Project description:Alternative splicing comprises a robust generator of mammalian transcriptome complexity. Splice site specification and activity are controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. A major subset of these interactions comprises interactions localized to the intronic U-rich polypyrimidine tract located immediately 5â to the majority of splice acceptors. alphaCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNP Es) comprise a subset of KH-domain proteins with high specificity and affinity for C-rich polypyrimidine motifs. Prior studies have revealed that binding of alphaCPs to C-rich motifs can modulate splicing and 3â processing of the human alpha-globin mRNA transcript in the nucleus as well as stabilization of the halpha-globin mRNA in the cytoplasm. In the current report, we demonstrate that alphaCPs have a positive impact on the activity of splice acceptor sites in a defined subset of mammalian transcripts via binding to polypyrimidine tracts that are predominantly C-rich. These findings lead us to conclude that the alphaCPs play a global role in determining the splicing activity and levels of cassette exon inclusion within the mammalian transcriptome. To test the impact of aCP proteins on alternative splicing, aCP proteins were knockdown from K562 cells by siRNA. Since aCP1 and aCP2 have redundent function, we therefore designed siRNAs capable of knockdown both isoform at the same time. 3 aCP1/2 combined knockdown and 3 control siRNA knockdown were performed in K562 cells. RNA-seq were then performed to identify alternative splicing pattern mediated by aCP proteins.

2016-03-01 | E-GEOD-71569 | biostudies-arrayexpress

Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

Project description:Alternative splicing comprises a robust generator of mammalian transcriptome complexity. Splice site specification and activity are controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. A major subset of these interactions comprises interactions localized to the intronic U-rich polypyrimidine tract located immediately 5’ to the majority of splice acceptors. alphaCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNP Es) comprise a subset of KH-domain proteins with high specificity and affinity for C-rich polypyrimidine motifs. Prior studies have revealed that binding of alphaCPs to C-rich motifs can modulate splicing and 3’ processing of the human alpha-globin mRNA transcript in the nucleus as well as stabilization of the halpha-globin mRNA in the cytoplasm. In the current report, we demonstrate that alphaCPs have a positive impact on the activity of splice acceptor sites in a defined subset of mammalian transcripts via binding to polypyrimidine tracts that are predominantly C-rich. These findings lead us to conclude that the alphaCPs play a global role in determining the splicing activity and levels of cassette exon inclusion within the mammalian transcriptome.

2016-03-01 | GSE71569 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)

Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.

2010-09-01 | GSE23514 | GEO

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