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. 2014 Jan 31;9(1):e87361.
doi: 10.1371/journal.pone.0087361. eCollection 2014.

A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events

Angela N Brooks  1 , Peter S Choi  1 , Luc de Waal  1 , Tanaz Sharifnia  1 , Marcin Imielinski  1 , Gordon Saksena  2 , Chandra Sekhar Pedamallu  1 , Andrey Sivachenko  2 , Mara Rosenberg  2 , Juliann Chmielecki  1 , Michael S Lawrence  2 , David S DeLuca  2 , Gad Getz  2 , Matthew Meyerson  3
Affiliations

A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events

Angela N Brooks et al. PLoS One. .

Abstract

Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35) have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA). Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML), in which U2AF1 is somatically mutated in 3-4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3' splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3' splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Splicing changes associated with U2AF1 S34F/Y mutation in lung adenocarcinoma and AML.
(A) Representation of somatic mutations in U2AF1 observed across 12 TCGA cancer types . Five cancer types had no somatic mutations in U2AF1. Amino acid positions are indicated. Zn, zinc finger; UHM, U2AF homology motif. (B) JuncBASE analysis of RNA-Seq data identified 131 and 369 splicing events significantly differentially spliced in lung adenocarcinoma and AML specimens bearing U2AF1 S34F/Y mutations, respectively.
Figure 2
Figure 2. Commonly altered splicing events associated with U2AF1 mutation in lung adenocarcinoma and AML.
A comparison of changes in splicing associated with U2AF1 somatic mutation in human cancers and U2AF1 S34F induction in HeLa cells.
Figure 3
Figure 3. Sequence preferences at altered 3′ splice sites associated with U2AF1 S34F/Y mutation.
(A) Proportion of altered cassette exon and alternative 3′ splice site events that show exon skipping versus exon inclusion. (B) Consensus sequence motifs identified at the proximal (prox 3′ss) and distal 3′ splice sites (dist 3′ss) of exon skipping events. (C) Consensus sequence motifs at the proximal and distal 3′ splice sites of exon inclusion events. Splicing changes of expressed and annotated 3′ splice site choices where the splice site choice is TAG vs. CAG or TAG vs. AAG in (D) HeLa cells+induced U2AF1 S34F or (E) HeLa cells+induced U2AF1 wild-type.
Figure 4
Figure 4. CTNNB1 3′ UTR splicing associated with U2AF1 S34F/Y mutation in lung adenocarcinoma and AML.
“Percent spliced in” (PSI) values of the proximal 3′ splice site of the CTNNB1 3′ UTR splice event in (A) lung adenocarcinoma and (B) AML. (C) RNA-Seq read coverage of the 3′ UTR event in HeLa cells with two U2AF1 non-induced controls, induction of U2AF1 wild-type, and induction of U2AF1 S34F.
Figure 5
Figure 5. Quantitative RT-PCR validation of splicing events affected by expression of U2AF1 S34F in 293T cells.
A fold difference between total gene expression and the inclusion isoform of each splicing event was normalized by the fold difference of a no-transfection control sample to yield a relative inclusion level for each sample. The genomic coordinates of tested splice events in (A) CTNNB1, (B) CHCHD7, and (C) PTBP1 are given in Table 1.
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