Details

Autor(en) / Beteiligte

• Selvanathan, Saravana P.
• Graham, Garrett T.
• Erkizan, Hayriye V.
• Dirksen, Uta
• Natarajan, Thanemozhi G.
• Dakic, Aleksandra
• Yu, Songtao
• Liu, Xuefeng
• Paulsen, Michelle T.
• Ljungman, Mats E.
• Wu, Cathy H.
• Lawlor, Elizabeth R.
• Üren, Aykut
• Toretsky, Jeffrey A.

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2015-03, Vol.112 (11), p.E1307-E1316

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2015

Link zum Volltext

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Significance Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from noncancer cells remains an ongoing challenge, and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anticancer targets. We determined that the key oncoprotein of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing that are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. 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.