CBTi-seq validates specialized alternative splicing patterns in gene-edited cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE304004 | GEO | 2026/01/01
REPOSITORIES: GEO
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Project description:CBTi-seq validates specialized alternative splicing patterns in gene-edited cells
| PRJNA1298739 | ENA
Project description:Splicing factors control exon inclusion in messenger RNA, shaping transcriptome and proteome diversity. Their catalytic activity is regulated by multiple layers, making single-omic measurements on their own fall short in identifying which splicing factors underlie a phenotype. Here, we propose that splicing factor activity can be estimated by interpreting changes in exon inclusion. To see if this is the case we benchmarked methods to construct splicing factor→exon networks and calculate splicing factor activity. Combining RNA-seq perturbation-based networks with VIPER (virtual inference of protein activity by enriched regulon analysis) accurately captures splicing factor activation modulated by different regulatory layers. This approach consolidates splicing factor regulation into a single score derived solely from exon inclusion signatures, allowing functional interpretation of heterogeneous conditions. As a proof of concept, we identify recurrent cancer splicing programs, revealing oncogenic- and tumor suppressor-like splicing factors missed by conventional methods. These programs correlate with patient survival and key cancer hallmarks: initiation, proliferation, and immune evasion. Altogether, we show splicing factor activity can be accurately estimated from exon inclusion changes, enabling comprehensive analyses of splicing regulation with minimal data requirements. In this dataset, we share our results from analyzing the BJ fibroblast-based model for carcinogenesis through proteomics and phosphoproteomics.
2025-07-27 | PXD066623 | Pride
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
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
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
Project description:Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we developed and methodically tested an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. Using the example gene RBFOX2, we validated protein-level changes and assessed the functional impact of isoform-specific knockdown. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all transcriptional and splicing events, including exon skipping and inclusion, alternative 5’ and 3’ splice sites, and alternative starts and ends.
2025-07-29 | PXD060902 | panorama
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.
2013-06-15 | E-GEOD-47945 | biostudies-arrayexpress
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
Project description:Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. siRNA knockdown of RBM38 was perfomed in human MCF-7 breast cancer cells. The efficiency of RBM38 knockdown was monitored by western blot using an RBM38 antibody (Santa Cruz Biotechnology, SC-85873). We conducted HJAY exon and exon junction array profiling on RNAs from four siRBM38 treated MCF-7 samples vs. four sicontrol treated MCF-7 samples Control / knockdown comparison.
2013-07-29 | E-GEOD-49293 | biostudies-arrayexpress
Project description:Background RNA sequencing (RNA-seq) is a powerful technique for identifying and quantifying transcription and splicing events, both known and novel. However, given its recent development and the proliferation of library construction methods, understanding the bias it introduces is incomplete but critical to realizing its value. Results Here we present a method, in vitro transcription sequencing (IVT-seq), for identifying and assessing the technical biases in RNA-seq library generation and sequencing at scale. We created a pool of > 1000 in vitro transcribed (IVT) RNAs from a full-length human cDNA library and sequenced them with poly-A and total RNA-seq, the most common protocols. Because each cDNA is full length and we show IVT is incredibly processive, each base in each transcript should be equivalently represented. However, with common RNA-seq applications and platforms, we find ~50% of transcripts have > 2-fold and ~10% have > 10-fold differences in within-transcript sequence coverage. Strikingly, we also find > 6% of transcripts have regions of high, unpredictable sequencing coverage, where the same transcript varies dramatically in coverage between samples, confounding accurate determination of their expression. To get at causal factors, we used a combination of experimental and computational approaches to show that rRNA depletion is responsible for the most significant variability in coverage and that several sequence determinants also strongly influence representation. Conclusions In sum, these results show the utility of IVT-seq in promoting better understanding of bias introduced by RNA-seq and suggest caution in its interpretation. Furthermore, we find that rRNA-depletion is responsible for substantial, unappreciated biases in coverage. Perhaps most importantly, these coverage biases introduced during library preparation suggest exon level expression analysis may be inadvisable. 5 rRNA-depleted samples with duplicates, 1 polyA selected, 1 total RNA, and 1 plasmid library all without replicates.
2014-05-21 | E-GEOD-50445 | biostudies-arrayexpress