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The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

ABSTRACT: Somatic mutations in the spliceosome have emerged in recent years as oncogenes in human cancer. These mutations are in the factors involved in splice site selection, including a missense mutation (Ser34Phe) in a conserved nucleic acid binding domain of the spicing factor U2AF1. This protein plays a critical role in recognition of the 3'-splice site and assembly of the pre-spliceosomal complex. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1. PAR-CLIP and RIP data show that U2AF1 directly binds mature mRNA in the cytoplasm and that binding on or near the start codon results in translational repression. This splicing-independent translational regulatory role of U2AF1 is altered by the S34F mutation, leading to elevated translation of hundreds of mRNA, as revealed by polysome profiling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE126912 | GEO | 2019/02/23

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

Multi-omics analysis of RNA binding, splicing and turnover alterations induced by mutant U2AF1 in myeloid malignancies.

Project description:Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA binding motifs; yet how they affect splicing and promote cancer remains unclear. The U2AF1/U2AF2 heterodimer is critical for 3’ splice site (3’SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure detecting contacts between U2AF1 and the 3’SS AG at single-nucleotide resolution (fractionated eCLIP-seq or freCLIP-seq). Our data reveal that U2AF1 S34F and Q157R mutants establish new 3’SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding (eCLIP-seq and freCLIP-seq), splicing (RNA-seq) and turnover (TimeLapse-seq or TL-seq) data, we predicted that U2AF1 mutations directly affect stress granule components. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies. Keywords: splicing, RNA binding, U2AF1, U2AF2, S34F, Q157R, hotspot mutations, 3'SS, 3' splice site, myeloid malignancies, eCLIP, freCLIP, RNA-seq, RNA turnover, TimeLapse-seq, TL-seq, RNA granules, stress granules, stress response, biomolecular condensates, MDS, AML

2022-03-17 | GSE195620 | 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

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

Identification of differentially spliced genes by wild type or S34F mutation of U2AF1

Project description:Identification of differentially spliced genes by wild type or S34F mutation of U2AF1 Examination of effects on splicing events by overexpressing wipdtype or S34F mutation of two U2AF1 isoforms in A549 cells. All experimental conditions are performed in duplicate.

2015-11-01 | E-GEOD-66553 | biostudies-arrayexpress

RNA-Seq data from U2 small nuclear RNA auxiliary factor 1 (U2AF1)-S34F and WT SKM-1 cells

Project description:Mutations in the U2 small nuclear RNA auxiliary factor 1 (U2AF1) gene are the common feature of a major subset in myelodysplastic syndromes (MDS). However, the genetic landscape and molecular pathogenesis of oncogenic U2AF1 S34F mutation in MDS are not totally understood. To understand how the cancer-associated U2AF1 S34F mutation promotes MDS, we next compared independent RNA samples of SKM-1 cells expressing wild-type U2AF1 and S34F mutant U2AF1 using RNA-sequencing (RNA-seq).Differentially expressed genes (DEGs) and significantly enriched pathways were identified by RNA-seq.

2021-03-13 | GSE166798 | GEO

U2AF1 mutations alter splice site recognition in hematological malignancies

2014-09-23 | GSE58871 | 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

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

RNA sequencing of mouse bone marrow cells from transgenic mice following short term induction of mutant U2AF1(S34F) or U2AF1(WT) expression concurrent with sudemycin D6 (or vehicle) treatment in vivo

Project description:Mutations in spliceosome genes occur in the bone marrow of approximately 50% of patients with myelodysplastic syndromes (MDS), with mutations in the splicing factor gene U2AF1 found in ~11% of MDS patients. We hypothesized that cells harboring a spliceosome gene mutation would have increased sensitivity to further perturbation of the spliceosome by splicing modulator drugs. To examine the effects of the splicing modulator drug sudemycin D6 on primary hematopoietic cells expressing mutant U2AF1(S34F), we treated transgenic mice expressing either mutant U2AF1(S34F) or U2AF1(wildtype, WT) concurrently with 5 days of sudemycin D6 treatment in vivo. We harvested bulk bone marrow cells 18 hours after the last day of treatment and performed strand-specific transcriptome sequencing to examine the cumulative pre-mRNA splicing changes associated with both mutant U2AF1 expression and sudemycin D6 treatment.

2016-11-15 | GSE89834 | GEO

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