Les oncoprotéines FET remodèlent l'épissage alternatif des sarcomes en détournant le complexe LASR

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 studied for the prototypical FET fusion EWSR1::FLI1, the main driver of Ewing sarcoma. Interestingly, RBFOX2, an RNA-binding protein (RBP) governing alternative splicing as part of a large assembly of splicing regulators (LASR) complex, has been identified as a key functional partner of EWSR1::FLI1. 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 when the expression of the corresponding fusion is prevented. This suggests that FET fusions inforce 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. As FET fusions lack canonical RNA-binding domains, we hypothesized that endogenous recruitment could be mediated indirectly via an RBP. By performing luciferase-based protein complementation assays, we demonstrated that almost all FET fusions could interact with RBFOX2. Surprisingly, RBFOX2 appeared to preferentially interact with the C-terminal domains of the fusions, which are derived from a wide range of unrelated TFs. RBFOX2 is known to function as part of a hetero-multimeric splicing complex called LASR. Based on our genome-wide splicing analysis analysis, we found that RNA-binding motifs of RBFOX2 and other members of the LASR complex were found enriched in the proximity of differentially regulated cassette exons. We further validated that members of LASR co-immunoprecipitated with FET fusions. Finally, we confirmed the importance of RBFOX2 and other LASR components in the modulation of splicing of endogenous and cancer-related gene targets of FET fusions. Altogether, our work provided evidence supporting a direct role in splicing for FET fusions by interacting with the LASR complex. Although the mechanisms by which the oncoproteins and LASR 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

Sorting and packaging of RNA into extracellular vesicles shape intracellular transcript levels.

peer reviewed[en] BACKGROUND: Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions. RESULTS: In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3' UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles. CONCLUSIONS: We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile

Le facteur de transcription oncogénique EWS-FLI1 conduit à l'instabilité de l'ARNm dans le sarcome d'Ewing

In most cases, Ewing sarcoma arises from a chromosomal translocation between the genes encoding a RNA-binding protein from the FET family (EWS) and a transcription factor from the ETS-superfamily (FLI1). Because the DNA-binding domain of FLI1 replaces the RNA-binding of EWS and is associated with a transactivation domain, the fusion protein EWS-FLI1 has early been viewed as an aberrant transcription factor. However, many evidences from litterature and our lab are challenging this view and led us to investigate a potential role in mRNA degradation for this oncoprotein.Contribution of mRNA decay to the oncogenic roles of FET-ETS fusion protein

Contribution de la dégradation de l'ARNm dans les fonctions oncogéniques d'un facteur de transcription de fusion

Gene fusions are an important class of somatic alterations in cancer. In many well-known cases, they encode aberrant fusion transcription factors (TFs) with neomorphic DNA-binding preferences. FET::ETS fusions represent a notable family of TFs. They result from the juxtaposition of a member of the FET (FUS/EWSR1/TAF15) family of RNA-binding proteins to a member of the ETS superfamily of TFs (e.g., FLI1 and ERG). These fusions are oncogenic drivers in many sarcomas and leukemias but challenging drug targets. An important limiting factor in developing therapies for them relies in our partial understanding of their underlying pathogenic molecular mechanisms. To date, the oncogenic functions of FET::ETS fusion proteins are almost exclusively confined to the control of mRNA synthesis. Based on a growing number of studies that identified non-canonical roles in the control of mRNA decay for various non-fusion (wild-type) DNA-binding TFs in human, we investigated whether FET::ETS fusion TFs might also be involved in mRNA decay. To test this possibility, we reasoned that Ewing sarcoma might represent an attractive model for a proof-of-concept study. Ewing sarcoma is an aggressive bone and soft-tissue childhood cancer as well as a paradigm for solid tumor development after a single genetic event. In ~85% of patients, this disease is driven by the fusion protein EWSR1::FLI1 (EF). Structurally, EF is a well-defined TF containing a potent amino-terminal transactivation domain that is intrinsically-disordered and a carboxy-terminal ETS DNA-binding domain. Molecularly, EF is known to orchestrate oncogenic gene expression programs by reprogramming enhancers and promoters via phase transition and hijacking of chromatin regulators; as well as by remodeling the 3D genome architecture. In this work, we report that EF also reprograms gene expression by affecting mRNA stability and decipher the molecular mechanisms underlying this function. We show that EF is recruited to mRNAs via interaction with the RNA-binding protein HuR (also known as ELAVL1), and promotes mRNA decay by binding to CNOT2, a component of the CCR4-NOT deadenylation complex. Interestingly, we evidence that EF antagonizes the normal mRNA protective function of HuR through its association with CCR4-NOT. Importantly, we show that EF-mediated mRNA decay supports Ewing sarcoma biology and yields a new vulnerability towards HuR inhibition. Finally, our data indicate that the control of gene expression by fusion TFs might represent a more complex scheme than anticipated, integrating mRNA synthesis and degradation, and thereby providing novel actionable molecular targets

Un nouveau regard sur le Sarcome d'Ewing: l'hypothèse que des fonctions de régulation post-transcriptionnelles de l'ARNm contribuent aux propriétés oncogéniques de la fusion EWS-FLi1.

The FET/TET proteins, which include TLS/FUS, EWS, and TAF15 are RNA- and DNA-binding proteins, with proposed functions in transcription and RNA processing. The family is notorious because it is involved in chromosomal translocations in which the 5' part of human FET genes is fused to the 3' region of genes encoding potent transcription factors, including members of the Erg transcription factor family (ERG, Fli1 and FEV). FET-Erg fusions have been associated with Ewing's sarcoma (ES) family tumors and acute myeloid leukemia and a plethora of evidence leaves little doubt that these fusions are essential to the development of these malignancies. Despite their importance, our knowledge on how FET-Erg oncogenic fusions impact on tumor biology remains incomplete. Because they include the C-terminal half of Erg proteins, which contains the ETS DNA-binding domain, fused to the amino-terminal portion of FET proteins, which behaves as a potent transcription activation domain, FET-Erg fusions have mostly been studied as oncogenic transcription factors. The current model suggests that FET-Erg fusions aberrantly regulate the transcription of genes that promote the establishment and/or the maintenance of the malignant phenotype. Despite years of investigations and identification of multiple transcriptional targets, this model has failed to fully decipher the oncogenic potential of FET-Erg fusions. In this project, we would like to take advantage of recent findings from our laboratories and assess the provocative hypothesis that important post-transcriptional functions of FET and Erg proteins, in particular in mRNA degradation and translation might be deregulated in FET-Erg fusions and contribute to their roles in cancer initiation and progression. We believe that a full understanding of oncogenic FET-Erg proteins functions can only arise from integrating their transcriptional and post-transcriptional functions. We are also convinced that this new perspective on FET-Erg fusions will provide novel therapeutic opportunities for ES and various FET-Erg-related cancers

Les fusions oncogéniques FET réorganisent les profils d'épissage alternatif pour induire la sarcomagenèse

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