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Transcription-dependent control of stem cell self-renewal and differentiation by the splicing factor U2AF1

ABSTRACT: Purpose: Here we describe the modulation of a gene expression program involved in cell fate. Methods: We depleted U2AF1 in human induced pluripotent stem cells (hiPSCs) to the level found in differentiated cells using an inducible shRNA system, followed by high-throughput RNAseq, revealing a gene expression program involved in cell fate determination. Results: Approximately 85% of the total raw reads were mapped to the human genome sequence (GRCh37), giving an average of 200 million human reads per sample for total RNA and 15 million human reads per sample for small RNA libraries. Conclusions: Our results show that transcriptional control of gene expression in hiPSCs can be set by the CSF U2AF1, establishing a direct link between transcription and AS during cell fate determination.

ORGANISM(S): Homo sapiens

PROVIDER: GSE119700 | GEO | 2018/12/03

REPOSITORIES: GEO

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Nanopore sequencing reveals isoform-specific alterations in human bronchial epithelial cells with U2AF1 S34F mutations

Project description:U2AF1 S34F is one of the most recurrent splicing factor mutations in lung adenocarcinoma (ADC) and has been shown to cause transcriptome-wide pre-mRNA splicing alterations. While U2AF1 S34F-associated splicing alterations have been described, the fate of affected mRNA isoforms remains largely unexplored. To better understand the impact U2AF1 S34F has on isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without U2AF1 S34F mutation. We find that nearly 75% (49,366) of our long-read constructed multi-exon isoforms do not overlap GENCODE or short-read assembled isoforms, a large proportion of which (12,397) are due to novel junction connectivity rather than novel splice site usage. We find 198 transcript isoforms with significant expression and usage changes relative to wild-type, some of which were not assembled by short-reads. We find an enrichment of isoforms from immune related genes are down regulated in the presence of U2Af1 S34F, none of which are observed to have splicing changes detected from long-read data. Finally, we reveal that isoforms likely targeted by nonsense-mediated decay are largely downregulated in U2AF1 S34F cells, suggesting that the impact of observed isoform changes may alter the translational output of affected genes. Altogether, our data builds on previous work by providing a higher resolution transcriptome snapshot of full-length isoform alterations associated with U2AF1 S34F in HBEC3kt cells.

2022-06-30 | GSE140734 | GEO

RNA sequencing of common myeloid progenitors following short term induction of mutant U2AF1(S34F) expression in transgenic mice

Project description:Mutations in the splicing factor gene U2AF1 have been found in the bone marrow of patients with myelodysplastic syndrome and acute myeloid leukemia, as well as in other cancers. To study the effects of mutant U2AF1(S34F) expression on hematopoiesis and pre-mRNA splicing in hematopoietic cells, we generated two inducible transgenic mouse lines expressing either mutant U2AF1(S34F) or U2AF1(wildtype, WT) as control. We performed strand-specific transcriptome sequencing on bone marrow common myeloid progenitor cells from U2AF1(S34F) and U2AF1(WT)-expressing mice to examine the pre-mRNA splicing changes associated with expression of mutant U2AF1(S34F). Donor-derived common myeloid progenitor cells from mice transplanted with U2AF1(S34F)/rtTA or U2AF1(WT)/rtTA bone marrow were sorted by flow cytometry, and RNA was extracted for transcriptome analysis. Ribosomal RNA was depleted prior to strand-specific RNA sequencing (TruSeq stranded library production, followed by 2 x 100bp paired-end sequencing performed on the HiSeq2000 platform from Illumina). Three samples were sequenced per genotype (n=3), and each sample was composed of bone marrow from 4-5 mice pooled. Reads were aligned to the mouse mm9 reference genome using TopHat (version 2.0.8), and we performed all subsequent analyses in R.

2015-05-11 | E-GEOD-66793 | biostudies-arrayexpress

U2AF1 mutations alter splice site recognition in hematological malignancies

Project description:Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 altered its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways implicated in myeloid disease such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in vivo, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1’s zinc finger domains. mRNA profiles of K562 cells expressing U2AF1 WT, mutants and knockdown of U2AF1 generated by deep sequencing.

2014-09-23 | E-GEOD-58871 | biostudies-arrayexpress

Isoform-specific depletion of U2AF1

Project description:RNA-Seq data of U2AF1 isoform depletion experiments.

2015-03-16 | E-MTAB-2682 | biostudies-arrayexpress

Next Generation Sequencing Facilitates Quantitative Analysis of MED23-knockdown and Control HUVEC Cells Transcriptomes

Project description:Purpose: RNA-seq analysis to evaluate the impact of MED23 knockdown on the transcriptome of HUVEC cells.Methods: Total RNA was isolated with TRIzol from Med23 knockdown HUVECs (n = 3) or control cells (n = 3). cDNA sequencing libraries were prepared with an NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The RNA-Seq FASTQ raw data were trimmed to remove low-quality reads and adapters using Trimmomatic. The trimmed reads were aligned to the mouse reference genome UCSC GRCh37/hg19 with HISAT2. Gene and transcript quantification was performed using Cutdiff. The results of the mapping of the RNA-seq reads, transcript assembly and abundance estimation were reported as fragments per kilobase of exon per million fragments mapped (FPKM).

2021-10-21 | GSE186112 | GEO

RNA sequencing of erythroid and granulomonocytic colonies differentiated from transduced bone marrow CD34+ cells expressing U2AF1 S34F mutation, U2AF1 wild-type or empty vector control

Project description:Mutations of the splicing factor U2AF1 are frequent in the myeloid malignancy myelodysplastic syndromes (MDS) and in other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and increasing bone marrow blasts. We investigated the impact of the common U2AF1 S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1 S34F expression in human hematopoietic progenitors impairs erythroid differentiation, and skews granulomonocytic differentiation towards granulocytes. RNA-sequencing of erythroid and granulomonocytic colonies revealed that U2AF1 S34F induced a higher number of cassette exon splicing events in granulomonocytic than erythroid cells, and altered mRNA splicing of many transcripts (expressed in both cell types) in a lineage-specific manner. The introduction of isoform changes identified in the target genes H2AFY and STRAP into hematopoietic progenitors recapitulated phenotypes associated with U2AF1 S34F expression in erythroid and/or granulomonocytic cells, suggesting a causal link. Importantly, we provided evidence showing that isoform modulation of the U2AF1 S34F target genes H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1 S34F MDS cells, raising the possibility of using splicing modulators therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity, and for the development of new targeted therapies.

2017-04-25 | GSE94153 | GEO

Mutant U2AF1-Induced Alternative Splicing of H2afy (macroH2A1) Regulates B-Lymphopoiesis in Mice

Project description:Acquired spliceosome gene mutations are among the most common genetic alterations in myelodysplastic syndromes (MDS). Here we present evidence that H2AFY(macroH2A1), a histone H2A variant, is a functional target that is alternatively spliced by mutant U2AF1(S34F), a spliceosome gene. Expression of H2AFY1.1, a H2AFY splice-isoform that is reduced by U2AF1(S34F) expression, rescues the reduction in B-cells observed in U2AF1(S34F) mice. Human MDS samples with U2AF1 mutations have a similar reduction in B-cells. Collectively, our data suggest that altered splicing of H2AFY contributes to MDS pathogenesis.

2021-08-31 | GSE125118 | GEO

Promoter R-loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing

Project description:R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells, and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor ChIP-seq datasets, we found that many splicing factors, including U2AF1 whose recognition of 3¢ splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We revealed that U2AF1 can bind directly to R-loops and DNA G4 structures at low nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1’s interaction with 3¢-splicing site, thereby modulating pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interaction of U2AF1 with these nucleic acid structures, and unveiled new functions of this interaction, i.e., promotion of phase separation of U2AF1 and modulation of mRNA splicing. Our work also established, for the first time, a biochemical basis for U2AF1’s occupancy in gene promoters.

2023-10-12 | GSE230254 | GEO

U2af1 is required for survival and function of hematopoietic stem/progenitor cells

Project description:U2AF1 is involved in the recognition of the 3’ splice site during pre-mRNA splicing. Mutations in U2AF1 are frequently observed in myelodysplastic syndromes. However, the role of wild-type U2AF1 in normal hematopoiesis has remained elusive. Using a novel conditional U2af1 knockout allele, we have found that deletion of U2af1 results in profound defects in hematopoiesis characterized by pancytopenia, ablation of hematopoietic stem/progenitor cells (HSPC) leading to bone marrow failure and early lethality in mice. U2af1 deletion impairs HSPC function and repopulation capacity. U2af1 deletion also causes increased DNA damage and reduced survival in hematopoietic progenitors. RNA sequencing analysis reveals significant alterations in the expression of genes related to HSC maintenance, cell proliferation and DNA damage response-related pathways in U2af1-deficient HSPC. U2af1 deficiency also induces splicing alterations in genes important for HSPC function. Collectively, these results suggest an important role for U2af1 in the maintenance and function of HSPC in normal hematopoiesis. A better understanding of the normal function of U2AF1 in hematopoiesis is important for development of appropriate therapeutic approaches for U2AF1 mutant induced hematologic malignancies.

2021-07-15 | GSE162888 | GEO

Whole transcriptome splicing analysis in isogenic lung epithelial and adenocarcinoma cell lines with or without a recurrent splicing factor mutation, U2AF1 (S34F)

Project description:The splicing factor gene, U2AF1, is recurrently mutated in a variety of human cancers, including lung adenocarcinomas. The most frequent U2AF1 mutant, U2AF1 p.Ser34Phe (S34F), induces specific changes in pre-mRNA splicing, but it is unclear how these splicing changes are regulated. We have used genomic editing methods to modify the U2AF1 gene locus in an immortalized human bronchial epithelial cell line (HBEC3kt) and in human lung adenocarcinoma cells with pre-existing U2AF1 alleles, creating a U2AF1 S34F allele in the endogenous locus of HBEC3kts and inactivating U2AF1 S34F alleles in two lung adenocarcinoma cell lines (H441 and HCC78). By comparing global splicing alterations in these isogenic pairs of cell lines, we have identified many splicing alterations that are associated with the U2AF1 S34F mutation. Further, by decreasing the levels of wild-type U2AF1 in the isogenic HBEC3kt cells, we show that the magnitude of mutant-associated splicing is proportional to the ratio of S34F:WT gene products. This observation suggest that wild-type U2AF1 is a negative regulator of splicing alterations induced by U2AF1 S34F.

2016-09-30 | GSE80136 | GEO

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