Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Abstract

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

Keywords: CLK1; EWS-FLI1; Ewing sarcoma; TERT; alternative splicing.

Fig. 1.

EWS-FLI1–interacting proteins are highly enriched in RNA processing. (A) GO classification for biological processes of MS-identified proteins. (B) KEGG pathway analysis of MS-identified proteins. (C) IPA network analysis shows the interactions between spliceosome proteins identified by MS (purple). Solid purple line indicates edges connecting proteins identified by MS. Transcriptional regulators (oval), enzymes (rhombus), complex (double circle), translation regulators (hexagon), transporters (trapezoid), and cytokines (square). IPA used direct (solid line) and indirect (dotted line) data to show predicted interactions between the proteins. Arrows represent regulation. (D) IPA-drawn MYC network showing EWS-FLI1–interacting proteins identified by MS and their direct and indirect interactions (key as in C). (E) IPA-drawn network shows role of RNA pol II in the interactome consisting of several proteins including enzymes, nuclear receptors, and transcriptional regulators identified by MS (key as in C). (F) Proteins validated by ELISA that directly bind to EWS-FLI1 are indicated by green shading, and their direct connection by green solid lines. Immunoprecipitated-validated indirect binding partners are in orange. MS identified proteins as potential partners but not validated by co-IP are in blue. Proteins predicted to occupy nodes in EWS-FLI1 but not identified by MS are in gray. Spliceosomal complex proteins are grouped and shaded based on published associations with spliceosomal subunits.

Fig. 2.

Exon expression patterns and overall gene expression are altered by EWS-FLI1. (A) Partek genome analysis of the exon array probe set shows relative exon-level expression across genes. Each point is the expression at a single probeset (nodes). The y axis indicates relative intensity of exon expression, and the x axis indicates each probeset in 5′ to 3′ direction. TC32 WT control, express EWS-FLI1 (red) is compared with shRNA EWS-FLI1 reduction (blue). To validate alternative splicing, qRT-PCR primers were prepared based on probesets showing AS (closed arrows). Open arrows indicate a region of the transcript of equal exon expression used for primer design to normalize intensity across each gene. (B) Partek analysis of exon expression in hMSC with EWS-FLI1 expression (red) and absence, control WT hMSC (blue) similar to A. (C) Cellular models of ES were evaluated for a gene set using qRT-PCR with primer identification based on examples in A. Across the top are five ES cell lines and hMSC ± EF. The gene list is shown in the first column. Those genes with alternative splicing similar to EWS-FLI1 are shown in green, no isoform switch in red, and inconclusive qRT-PCR in light orange.

Fig. 3.

Alternatively spliced genes by EWS-FLI1 impact diverse cellular processes. (A) Comparison of de novo transcriptome reconstruction of TC32 WT, shRNA EWS-FLI1, and YK-4–279 treatment. Classification of alternative splicing events includes skipped exons (SE), retained introns (RI), mutually exclusive exons (MXE), alternative 5′ splice sites (A5SS), and alternative 3′ splice sites (A3SS). Each type of event is quantified as a percentage of the top six events. The top six events in each sample type constitutes ∼80% of all splicing patterns found in each sample, with the remaining 20% being more complex versions of these five basic types. (B) Exon-centric RNA-seq coverage maps of exons affected by EWS-FLI1. The numbers of exon junction reads are indicated adjacent to the connecting bars of the coverage map. Schematic and exon-centric coverage maps showing read depth [reads per kilobase of transcript per million reads mapped (RPKM)] for CLK1, CASP3, and PPFIBP1. Exon annotation was derived from GRCh37 annotation. Mixture of Isoforms (MISO) was used to estimate PSI values for each annotated event. Each event has associated 95% confidence intervals. On the right, PCR validation gels associated with each splicing event were used to calculate the PSI, shown below each lane. (C) Multiple EM for Motif Elicitation (MEME) was used to generate a motif from CLIP reads. (D) Sequence regions ± 125 bp from exon–intron junctions at 5′ and 3′ ends were searched for occurrences of this motif (red). A random motif was generated for comparison of occurrence by chance (blue). The peak shows high-frequency localization of EWS-FLI1 at the 5′ intron–exon boundary. (E) RNA secondary structure within a 200-bp window was based on the minimum free energy (MFE) which was calculated for all CLIP reads sequenced (red). A null set was generated by randomly sampling exonic regions of the same length as the sequenced reads (blue). (F) PCR products of alternatively spliced exons of CLK1, CASP3, and PPFIBP1 based on shRNA reduction of DDX5, hnRNP K, and PRPF6 in TC32 cells with their corresponding scrambled controls in alternating lanes. The PSI of exon inclusion is shown below each lane.

Fig. 4.

TERT is alternatively spliced to generate an isoform with enhanced telomerase activity. (A) Isoform-specific TERT semiquantitative RT-PCR primer locations are indicated by the forward and reverse arrows based on exon array analysis. The exons are indicated by gray boxes and exon-specific splice changes of skipped exon by the green line. (B) TC32 ± EWS-FLI1 mRNA semiquantitative RT-PCR products using primers shown in A. The PSI of exon 11 inclusion are shown below each lane. (C) Schematic of the TERT minigene (MG), indicating exons 10, 11, and 12 including 300 bp on the either side of the exon–intron junction regions of introns 10 and 11. Minigene-specific PCR primer locations are indicated by forward and reverse arrows. (D) Minigene (MG) plasmids were transfected into TC32 WT or shRNA EWS-FLI1–reduced cells. PCR products using MG-specific primers are shown. (E) Exon-centric coverage map for TERT was generated from deep sequencing described (Fig. 3B). (F) RNA immunoprecipitation (RIP) showing the presence of TERT mRNA bound to EWS-FLI1 compared with total 18S vs. IgG control IP. (G) Telomerase activities were analyzed by TRAP assay. TC32 WT, shRNA EWS-FLI1, shRNA hTERT with or without re-expression of WT TERT, or expression of γ-TERT were analyzed in real-time TRAP assays.

Fig. 5.

YK-4–279 treatment of ES cells reverts EWS-FLI1–modulated alternative splicing changes and does not mimic transcriptional effects of EWS-FLI1 reduction. (A) A qRT-PCR gene expression panel for canonical EWS-FLI1 transcriptional targets compared WT TC32, EWS-FLI1 reduction (ΔEF, blue bars), and YK-4–279 treatment (orange bars). Graph shows relative fold expression calculated using ΔΔct value changes from WT (set at 1.0). *P = 0.04 (NR0B1), 0.001 (p57), 0.003 (hTERT), 0.03 (Id2), 0.03 (EZH2), 0.006 (UPP1), 0.02 (GLI1), and 0.003 (PTPL1). (B) EWS-FLI1 was immunoprecipitated following treatment with YK-4–279 or DMSO control in TC32 cells for 15 h. The total nuclear lysate (TNL) treated with YK-4–279 was used as input control (lane 1), FLI1 antibody (lanes 2 and 4), and rabbit polyclonal IgG antibody (lanes 3 and 5). Antibodies for specific protein detection are shown to the right of each panel. (C) Alternative splicing screen comparing YK-4–279 and doxorubicin treatment using the same exon array identified RT-PCR primers as in Fig. 2C. Genes with EWS-FLI1 exon-specific isoform switch, similar to EWS-FLI1, are green, and no isoform switch is red. (D) Schematic and exon-centric coverage maps (RPKM) for CLK1, CASP3, and PPFIBP1 derived from deep sequencing (key as in Fig. 3B). MISO was used to estimate PSI values for each annotated event, and 95% confidence intervals are shown. WT TC32 data are red, and YK-4–279 treatment is orange. To the right, PCR validation gels show the splicing change, and the PSI is calculated from densitometry and shown beneath each graph. (E) Elongation rates for 10 min for the NR0B1 gene following 5,6 dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) removal are shown for WT TC32 cells (scrambled, Upper two panels) and for EWS-FLI1–reduced TC32 cells (shEWS-FLI1, Lower two panels). Polymerization rate was calculated following bromouridine (BRU) incorporation from the time of DRB washout. The second and fourth panels care concomitantly treated with YK-4–279 during the elongation period. Read depth (RPKM) calculated on y axis is based on immunoseparation of BRU-labeled message followed by deep sequencing. NROB1 transcript was derived from GRCh37 annotation shown at the bottom.

Fig. 6.

Alternative splicing patterns in ESTS and TC32 have similar exon-specific expression. (A) Gene-normalized probeset intensity plots for NC and hMSC WT (−EF) with expression of EF (+EF) as well as TC32 cells as WT (+EF) and shRNA reduction of EWS-FLI1 (−EF), and ESTS. Each probeset has been normalized by gene expression to provide a relative measure of exon inclusion. (B) PCA plots based on the differential exon expression leading to alternative splicing profiles for patients with localized ES. Patients who did not experience recurrence (open triangle, black) and patients who experienced recurrence (closed circle, red) are plotted in the first three principal component dimensions. Variance in each principal component is listed along the axes. (C) Dendrogram clustering of first three principal components evaluating patients with localized disease illustrating weak grouping effect separating patients with (red) and without (black) recurrence (P = 0.1). (D) PCA plots based on the differential exon expression leading to alternative splicing profiles for patients with metastatic ES. Patients who did not experience recurrence (open triangle, black) and patients who experienced recurrence (closed circle, red) are plotted in the first three principal component dimensions. Variance in each principal component is listed along the axes. (E) Dendrogram clustering of the first three principal components evaluating patients with metastatic disease illustrating strong grouping effect separating patients with (red) and without (black) recurrence (P = 0.05).