Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.
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.
Department of Oncology and Pediatrics, Georgetown University, Washington, DC 20057;
Saravana P
SP
Selvanathan
Department of Oncology and Pediatrics, Georgetown University, Washington, DC 20057;
Garrett T
http://orcid.org/0000-0002-1097-0962
GT
Graham
Department of Oncology and Pediatrics, Georgetown University, Washington, DC 20057;
Hayriye V
HV
Erkizan
Department of Pediatric Hematology and Oncology, University Hospital Münster, Westfalian Wilhelms University Münster, 48149 Münster, Germany;
Uta
U
Dirksen
Protein Information Resource, Georgetown University and University of Delaware, Washington, DC 20057;
Thanemozhi G
TG
Natarajan
Department of Pathology, Center for Cell Reprogramming, Georgetown University, Washington, DC 20057; and.
Aleksandra
A
Dakic
Department of Pathology, Center for Cell Reprogramming, Georgetown University, Washington, DC 20057; and.
Songtao
S
Yu
Department of Pathology, Center for Cell Reprogramming, Georgetown University, Washington, DC 20057; and.
Xuefeng
X
Liu
Department of Radiation Oncology and Translational Oncology Program and.
Michelle T
MT
Paulsen
Department of Radiation Oncology and Translational Oncology Program and.
Mats E
ME
Ljungman
Protein Information Resource, Georgetown University and University of Delaware, Washington, DC 20057;
Cathy H
CH
Wu
Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan, MI 48109.
Elizabeth R
ER
Lawlor
Department of Oncology and Pediatrics, Georgetown University, Washington, DC 20057;
Aykut
A
Üren
Department of Oncology and Pediatrics, Georgetown University, Washington, DC 20057; jat42@georgetown.edu.
Jeffrey A
JA
Toretsky
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics; D006801:Humans; D007211:Indoles; D007438:Introns / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034802:RNA-Binding Protein EWS / Q000235:genetics / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000235:genetics / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism
C106031:EWS-FLI fusion protein; D007211:Indoles; D015514:Oncogene Proteins, Fusion; D020033:Protein Isoforms; D051789:Proto-Oncogene Protein c-fli-1; D012333:RNA, Messenger; D034802:RNA-Binding Protein EWS; C562345:YK 4-279; C509186:TERT protein, human; D019098:Telomerase
CLK1; EWS-FLI1; Ewing sarcoma; TERT; alternative splicing