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, 41 (8), 733-741

Integrative Profiling of Alternative Splicing Induced by U2AF1 S34F Mutation in Lung Adenocarcinoma Reveals a Mechanistic Link to Mitotic Stress

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Integrative Profiling of Alternative Splicing Induced by U2AF1 S34F Mutation in Lung Adenocarcinoma Reveals a Mechanistic Link to Mitotic Stress

Suyeon Kim et al. Mol Cells.

Abstract

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.

Keywords: S34F; U2AF1; alternative splicing; lung adenocarcinoma; mitotic stress.

Figures

Fig. 1
Fig. 1. KD and mutant expression lead to distinct AS events
(A) Immunoblot showing KD of endogenous U2AF1 and expression of exogenous wt and S34F mutant U2AF1. Ctrl indicates control empty vector virus infection. (B) Venn diagram of genes showing AS upon KD or mutant expression. Note the limited overlap between KD and mutant expression in contrast to expression of the two mutants. (C–F) Representative cases of AS from the four indicated gene set categories. The alternatively spliced exon and surrounding exons are diagrammed. Arrows indicate oligonucleotide primers used for RT-PCR shown below. To the right are RNA-seq read coverage of the diagrammed exons. Directions of transcription are indicated above. Note the similarity in AS induced by the two U2AF1 splice variants.
Fig. 2
Fig. 2. Sequence preferences at altered 3′ splice sites associated with U2AF1 S34F mutant expression or KD
(A) On the left side, various types of alternative splicing (AS) events are schematically shown. Exons and introns are represented as blocks and dotted lines respectively. On the right side, the counts of AS events associated with U2AF1 S34F expression or KD are tabluated (FDR < 0.01). (B) On the left side, exon exclusion and inclusion are shown schematically for SE and A3SS. Wild type splicing is indicated by black lines above the genes and AS induced by mutant expression or KD are shown in red below. Tabluation shows that most AS events induced by mutant expression or KD are in fact exon exclusion. (C) The sequences near the 3′ splicing site are shown. Note that nucleotide preference at the −3 position is changed from T to C/A upon mutant expression. Sequence logos are obtained using WebLogo (http://weblogo/berkeley.edu/loco/cgi).
Fig. 3
Fig. 3. Integrated profiling of alternative splicing with LUAD patient data and GO analysis
(A) Venn diagram of genes that have undergone AS from KD, from mutant expression and in LUAD cases with S34F mutation. Gene lists are presented for intersection gene sets. (B) GO terms and genes from gene set enrichment analysis with DAVID on 47 AS events found in the intersection of LUAD patient set and mutant expression set. Note that no enriched term was found for genes from intersection of LUAD patient set and KD set.
Fig. 4
Fig. 4. KD and mutant expression lead to mitotic stress at distinct stages
(A) Flow cytometric analyses of cell cycle progression. Note that both KD and mutant expression induce G2/M arrest. Results are average ± standard error of the mean (SEM) of three independent assays. (*) represents P-value of < 0.05 compared to control condition. (B) Representative images of cells after KD and mutant expression in the affected sub-stages. Note that after KD, centrosomes are not separating even though chromosomes are condensed. Also note that not all chromosomes (white arrows) are aligned in the center after mutant expression during metaphase. (B, C) Mitotic sub-stage distribution of cells after KD and mutant expression. Cells with condensed chromosomes with microtubules extending from centrosome are grouped together as prophase/prometaphase cells. Cells with centrosomes in opposite ends with chromosomes aligned in the middle are considered metaphase cells. Cells showing mitotic spindle-mediated chromosomal migration are considered anaphase cells. Note that KD results in accumulation of cells in prophase/prometaphase and decrease in fractions of cells in all subsequent stages while mutant expression increases the fraction of cells in metaphase and decreases those in anaphase and telophase. Results are average ± SEM of three independent assays. (*) represents P-value of < 0.05 compared to control condition. (D) Fractions of cells with monopolar spindles after KD and of cells with misaligned chromosomes after mutant expression (b and a) are shown. Results are average ± SEM of three independent assays. (*) represents P-value of < 0.05 compared to control condition.
Fig. 5
Fig. 5. Rebound proliferation of U2AF1 S34 mutant-expressing A549 cells
(A) 2 days after infection, the trypsinized cells were plated in 96-well plate, and cell proliferation was examined for upto 15 days. Note that U2AF1 S34 mutant-expressing cells grew in a robust manner after initially stunted cell division. (B) U2AF1 S34 mutant-expressing cells grew via clonal expansion. Virus-transduced cells were cultured for 15 days in 35mm plates. Note the clonal expansion of S34F mutant -expressing cells after presumptive apoptosis of most of the cells.

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