Abstract :
[en] The FET (FUS, EWSR1, TAF15) genes are commonly involved in chromosomal translocations resulting in their fusion with various transcription factors (TF) genes. These genomic abnormalities are hallmarks of several sarcomas and leukemias, and are found along few other alterations in these neoplasms. The chimeric proteins encoded by these fusion genes share a similar architecture, with a strong aminoterminal transactivation domain derived from FET proteins, and a carboxyterminal DNA-binding domain derived from the TF partner. As this structure is reminiscent of that of a TF, the oncogenic potential of FET fusion proteins has been first attributed to their ability to reprogram transcription. However, this transcriptional role is not sufficient to fully explain how these oncoproteins single-handedly drive various cancers. Indeed, growing evidence points towards novel post-transcriptional roles for FET fusions, notably in the alternative splicing of pre-mRNA. Such a function has previously been demonstrated for the prototypical FET fusion EWSR1::FLI1, the main driver of Ewing sarcoma.
In this project, we aim to determine whether the pre-mRNA alternative splicing function observed for EWSR1::FLI1 could be a shared mechanism promoting FET fusion-driven oncogenesis. RNA-sequencing of various FET-translocated sarcoma cell lines showed that thousands of alternative splicing events are induced upon FET fusion depletion. This suggests that FET fusions enforce a specific splicing landscape in their corresponding sarcoma. In addition, we showed that a representative panel of FET fusions promoted exon inclusion of a reporter minigene, but only when directly tethered onto its pre-mRNA, suggesting that the control of splicing by FET fusions might be direct and might rely on its recruitment onto pre-mRNA. The association of FET fusions to RNA was subsequently confirmed in several sarcoma cell lines, and we established that their recruitment might be mediated by intermediary RNA-binding proteins. In line with this hypothesis, the binding motifs of numerous splicing factors were found to be significantly enriched in regions flanking FET fusion-regulated exons. The interaction of FET fusions with several of these proteins was confirmed through a luciferase-based protein complementation assay and endogenous coimmunoprecipitation experiments. Finally, we found several splicing events to be controlled by all FET fusions. To test the biological relevance of isoforms favored by FET fusions, we prevented their expression and assessed the impact of this depletion on sarcoma cells proliferation and phenotype.
Altogether, our work provided evidence supporting a direct role in splicing for FET fusions by interacting with splicing factors. We also suggested that FET fusion-regulated splicing events play biologically relevant roles in FET fusion-mediated sarcomagenesis. Although the mechanisms by which the oncoproteins and splicing factors collaborate to promote oncogenesis remain unclear, we have established a moonlighting function for these driver fusion proteins that could be crucial in our understanding of the tumorigenesis of multiple neoplasms.
