Global PAS-seq (polyA-site-sequencing) profiling in control and CFIm25 KD human embryonic stem cells (H9 cell line)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE176343 | GEO | 2022/04/21
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: To identify all of the APA targets of CFIm25 on a global scale and develop an algorithm that can idenitify APA events from standard RNA-seq data Methods: RNA from HeLa cells treated with control siRNA and CFIm25 siRNA were subject to RNA-Seq. Using a custom-designed algorithm to mine RNA-seq data for novel APA events regulated by CFIm25. Results: We identified over 1,400 genes with shortened 3’UTRs after CFIm25 knockdown. Importantly, we show that as a consequence of APA, many of these mRNAs have greatly enhanced protein expression due to the loss of destabilizing features within the 3’UTR. Conclusions: Our study underscored the critical function of the CFIm complex members in governing APA and establish a previously unknown link between APA and metabolic pathways important for tumor progression. Hela cell line mRNA profiles of control treated and CFIm25 Knockdown were generated by RNA-Seq using Illumina GAIIx.
2014-05-15 | E-GEOD-42420 | biostudies-arrayexpress
Project description:Systemic sclerosis (SSc - scleroderma) is a multi-system, fibrotic disease affecting skin and internal organs. Alternative polyadenylation (APA) involves the differential usage of polyadenylation signals, which gives rise to transcripts with variable 3'UTR length. The mammalian Cleavage factor I 25kDa subunit (CFIm25, encoded by NUDT21) is a key regulator of alternative polyadenylation APA and its depletion causes predominantly 3'UTR shortening through loss of stimulation of distal polyA sites resulting in increased used of proximal polyA sites. A shortened 3'UTR will often lack microRNA target sites, resulting in increased mRNA translation due to evasion of microRNA-mediated gene repression. Herein, we report that CFlm25 is downregulated in SSc skin, and primary dermal fibroblasts, andas well as in two murine models of dermal fibrosis.The purpose of this experiment is to identify the APA targets of CFIm25 in human skin fibroblasts. Following the knockdown of CFIm25 in normal human skin fibroblasts, we identified 971 genes with shortened 3’UTRs, including those involved in the transforming growth factor-beta signaling pathway.
2019-09-11 | GSE137276 | GEO
Project description:Alternative polyadenylation (APA) is a regulatory mechanism that controls gene expression level and function through the usage of different transcription termination sites, but how APA is regulated to influence epidermal tissue differentiation remains under-characterized. Leveraging 3’READS+, we identified prevalent APA in keratinocytes. We further identified a high-confidence list of genes that alters their usages of polyadenylation site (PAS) during keratinocyte differentiation. To begin to understand the mechanisms that modulates PAS selection within a given gene, we identified that the expression of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is down regulated during keratinocyte differentiation. Suppression of CPSF using RNAi or CRISPRi strongly impaired epidermal regeneration with upregulation of terminal differentiation marker gene expression. Mechanistically, we identified that CPSF suppression decreased the usage of the GRHL3 proximal PAS and upregulated full-length GRHL3 mRNA production. Knockdown of GRHL3 in the context of CPSFi partially restored differentiation marker gene expression. We also found CPSF interacts with RNA-binding proteins to control PAS usage. Thus, our data suggest a model where CPSF binding preference and PAS choices regulate epidermal terminal differentiation.
2020-11-02 | GSE127223 | GEO
Project description:Purpose: To identify all of the APA targets of CFIm25 on a global scale and develop an algorithm that can idenitify APA events from standard RNA-seq data Methods: RNA from HeLa cells treated with control siRNA and CFIm25 siRNA were subject to RNA-Seq. Using a custom-designed algorithm to mine RNA-seq data for novel APA events regulated by CFIm25. Results: We identified over 1,400 genes with shortened 3’UTRs after CFIm25 knockdown. Importantly, we show that as a consequence of APA, many of these mRNAs have greatly enhanced protein expression due to the loss of destabilizing features within the 3’UTR. Conclusions: Our study underscored the critical function of the CFIm complex members in governing APA and establish a previously unknown link between APA and metabolic pathways important for tumor progression.
2014-05-15 | GSE42420 | GEO
Project description:We carried out CFIm25 KD experiments in H9 cell line (hESCs). After poly(A+)-seq, we found the expression of hundreds of genes were changed. To investigate if the effect is a direct or indirect effect of CFIm25 KD, we next carried out 3Flag-CFIm25 overexpression in CFIm25 KD cells for gene-rescue experiments. After we obtained this cell line, here we performed poly(A+)-seq in control H9 and (CFIm25 KD+ 3Flag-CFIm25 overexpression)H9 cell lines. The goal of this experiment is to investigate if CFIm25 overexpression could rescue the gene expression phenotype caused by CFIm25 KD.
2022-04-21 | GSE176328 | GEO
Project description:We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.
2019-08-14 | PXD014842 | Pride
Project description:Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. We first analyzed HeLa cell transcriptome using PAS-Seq to demonstrate that PAS-Seq not only accurately and comprehensively identifies poly(A) junctions, but also provide quantitative information on the relativel abundance of APA isoforms. Next we analyzed the mouse embryonic stem cells, neural stem/progenitor cells, and neurons by PAS-Seq to characterize the dynamic changes of mRNA polyadenylation during stem cell differentiation.
2011-02-26 | E-GEOD-25450 | biostudies-arrayexpress
Project description:To understand how CFIm25 associates with genomic DNA during co-transcriptional mRNA processing,we carried out ChIP-seq analysis for CFIm25 in control and CFIm25 KD cells (CFIm25 KD cells as normalization background)
2022-04-21 | GSE178076 | GEO
Project description:We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.
2020-04-27 | GSE135384 | GEO
Project description:We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.
2020-04-15 | GSE142681 | GEO