Functional Interactions in Transcription and Splicing of Ewing’s Sarcoma

Ewing’s sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA and DNA-binding proteins with unknown cellular role. EWS protein is encoded by the EWS oncogene on chromosome 22q12, a target of chromosomal translocations in Ewing’s sarcoma tumors. The exact mechanism of EWS participation in gene expression and pathogenesis of the resulting cancers is not defined. The binding partners of native EWS and EWS fusion proteins (EFPs) are described schematically in a model, an attempt to link the transcription with the splicing. The experimental data about the partnerships of EWS and EFPs are summarized, which may lead to better understanding of their function.


Introduction
Transcription and splicing seems to be connected by proteins with roles in both processes, coordinating them, such as TET proteins, since their NTDs mediate interactions with RNA polymerase II (Pol II), while their CTD binds to splicing factors.Thus TET proteins may recruit splicing factors to the Pol II, which coordinates pre-mRNA processing events [1].
The participation of native Ewing's sarcoma protein (EWS) and its oncogenic fusion proteins (EFPs), as well as their reported binding partners in transcription and splicing, could be described schematically by a model to link the transcription with the splicing in ESFTs.Some EWS and EWS/FLI1 interacting partners (including Pol II subunits) are implicated in transcriptome and spliceosome and are directly involved in mRNA synthesis or splicing.
1.1.Ewing's Sarcoma.Ewing's sarcoma, first described by James Ewing in 1921, is still a cryptic malignancy.Ewing's sarcoma family tumors (ESFTs) afflict children and young adults, encompassing Ewing's Sarcoma of bone, extraosseous (soft-tissue) Ewing's sarcoma, primitive neuroectodermal Tumor (PNET), and Askin's tumor.ESFTs have high propensity to metastasize in bone, bone marrow, and lung.ESFTs are aggressive round cell tumors of putative stem cell origin, for which prognostic biomarkers and novel treatments are needed [2,3].ESFTs are chemotherapy-sensitive cancers, and even patients with metastatic disease commonly achieve remission.Diagnosis of Ewing tumor is based on pathologic and molecular findings [4].The EFPs are promoter-specific transcriptional activators, due to EWS-activation domain (EAD) and a DNA-binding domain (DBD) from the fusion partner.About 85% of Ewing tumours carry the most frequent EWSR1/FLI1 fusion gene that is critically important for maintaining the tumor phenotype of the disease.The EWS/FLI1 may be fully transforming only in a mutated cell that possesses additional genetic alterations, supported by the experiments with limb transgenic mice and cell culture.The mutations occur after the t(11; 22) translocation occurs (Figure 1), but it is also possible that EWS/FLI1 transforms a cell that carries a silent and critical mutation in another gene [5].EWS-deficient mice demonstrated that EWS is required for the completion of B cell development and meiosis [6]. KO mice displayed aging-like phenotypes, and both tumorigenesis and senescence are intimately tied to stem cells.The loss of endogenous  results in dramatic changes in the dynamics of hematopoietic stem progenitor cells and a progressive and severe postnatal atrophy of  −/− mice hematopoietic organs. −/− cells display impaired DNA repair capacity and show a diminished capacity to sustain survival of lethally irradiated animals in serial transplantations. may play a role in DNA repair and/or DNA recombination [7].

Native EWS.
The EWS is a member of the TET family of RNA and DNA-binding proteins.EWS is located in the nucleus, associated with components of the basal transcription, RNA-splicing factors [8] and G-protein coupled receptor signaling, and relocates from cytoplasm to ribosomes upon Pyk2 activation [9].EWS consists of an N-terminal transcriptional activation domain (TAD, EAD, NTD, aminoacids (AA) 1-264) and a C-terminal RNA-binding domain (RBD, CTD), extensively methylated at arginine residues, and containing a putative zinc-finger domain [10,11].
The EAD is intrinsically disordered and resembles many chromatin organizing proteins [12,13].Multiple Tyr residues are essential for EAD function [12].All three RGG motifs of EWS participate in self-association required for the EWS nuclear import [14].

EWS/ETS Fusions.
The generation of chromosomal translocations in Ewing's sarcoma could be mediated by a mechanism of illegitimate recombination, occurring before interchromosomal joining [17].The resulting EFPs are potent transcriptional activators that interact with other proteins required for mRNA biogenesis.EFPs induce tumorigenesis by perturbing the gene expression and participate in signaling cascades required for oncogenesis [18].
Specific chromosomal translocations fuse EWS to ETS family members (FLI, ERG, FEV, ETV1, and ETV4) in Ewing's sarcomas to give different isoforms of EWS/ETS oncogenic fusions, where the EAD is fused in-frame to the DNA-binding domain (DBD) of the corresponding ETS protein (Figure 1).Around 85% of Ewing's tumours carry the most frequent EWSR1/FLI1 fusion.Phosphorylation, acetylation, and glycosylation are posttranslationally modifying mechanisms, affecting EWS/FLI1 activity.Self-association of EWS and EWS/FLI1 and interaction of EWS/FLI1 with EWS and FLI1 were observed in vivo, involving the N-terminal and centrally localized amino acids [19].The EWS/WT1 phosphorylation on Ser and Tyr is affecting the DNA binding and homodimerization [20].The transactivation by EWS/ATF1 does not require dimerization, while the bZIP domain is necessary for dimerisation and DNA binding [21].
EWS/FLI1 is acting as a strong transcriptional activator and, in contrast to wt FLI1, is a potent transforming agent [22].EWS/FLI1 induces gene expression changes by causing smaller alterations in multiple stages of the gene regulation and modulating both transcript synthesis and mRNA degradation.EWS/FLI may have DNA-binding-independent function, blocking the normal function of wt EWS expression [18].The flexible and unfolded conformation allows EFPs to bind many interacting partners.Direct or indirect target genes of EWS/FLI contribute to tumor growth and progression, such as IGFBP3, GSTM4, CDKN1A, TGFBRII, VEGF, CAV1, E2F8, FOXO1, and NFKBIL2 [23,24].The "core" regulators, including genes NR0B1, NKX2.2, and GLI1, are essential for oncogenic transformation in Ewing's sarcoma [18].EWS/FLI1 repressed miRNAs are targets in IGF signalling pathway, a pivotal driver of Ewing's sarcoma oncogenesis [25].Both EWS/FLI1 and FLI1 act as antiapoptotic agents, targeting the CBP/p300 pathways in vitro and in vivo [26].

Native EWS in Transcription and Splicing of Normal
Cells.EWS contains an IQ domain that is phosphorylated by protein kinase C (PKC) and is interacting with calmodulin (CaM), a regulatory link between Ca 2+ signal transduction pathways and RNA processing [27].The EWS region interacting with ZFM1 (transcriptional repressor identical to splicing factor SF1) maps to 37 AAs within its NTD (Figure 1).The TET proteins are present in distinct TFIID populations, associated with Pol II holoenzyme, and cooperate with ZFM1 in mRNA processing during transcript elongation [28].The EAD binds to the coactivator CREB-binding protein, which is implicated in chromatin remodeling, and the hypophosphorylated Pol II [29].Full-length EWS forms multifunctional complexes with Pol II and TFIID that may be physiologically relevant [15].EWS interacts with Pol II subunits via hsRpb3.The AAs (1-82) of EWS possess full transforming activity and interact with Pol II.TBP dimerization inhibits the DBD and regulates kinetics of TBP-DNA interaction [30].EAD binds hRpb5 and directly Rpb7 by yeast two-hybrid screening, coimmunoprecipitation, and pull-down assays [15,31,32].Rpb7 and Rpb4 are required for EAD-mediated transactivation in yeast [33].Overexpression of recombinant hsRPB7 specifically increases the gene activation by EWS-chimeric TFs [31].The prolonged hsRpb4/7 depletion is cytotoxic, revealing a gene-specific role in human cell viability [34].
Various genotoxic stress inducers (camptothecin (CPT)) inhibit the interaction between EWS (Pol II-associated factor) and YB-1 (spliceosome-associated factor), resulting in cotranscriptional skipping of MDM2 gene exons, encoding the main p53 ubiquitin ligase.Disruption of the EWS, and YB-1 interaction by CPT may alter the local recruitment of the splicing machinery, affecting MDM2 exon splicing [35].Proposed is a communication between the transcriptional and splicing machineries mediated by Rpb7, EWS and YB-1 proteins [35].Proposed are TLS modulation mechanisms of YB-1-directed gene expression of MMP-1 gene, involved in tumor invasion and metastasis, where TLS may be loaded onto nascent transcripts at the promoter, integrating transcript processing and translation [16].MALDI-MS identified more than 70 prespliceosome-associated proteins, including U1 and U2 snRNP proteins, non-snRNP splicing factors, DEAD-box protein p68, RHA, YB-1 and TLS [36].
Recombinant TAF15 and U1C directly interact in vitro (required is NTD of TAF15) [37].EWS interacts via its RBD with RNase-sensitive protein complexes, consisting mainly of hnRNPs and RNA helicases.HnRNP (M and U), RNAhelicases (p68 and p72), actin, and tubulin interact directly with EWS.EWS and p68 or p72 colocalize in the nucleus of HEK cells [38].EWS depletion results in alternative splicing changes of genes ABL1, CHEK2, MAP4K2 involved in DNA repair and genotoxic stress signaling [39].EWS interacts with both the CREB-binding protein (CBP) [26,29] transcriptional coactivator and also the transcriptional repressor SF1 [28], positively and negatively regulating transcription [1].RGG domain of EWS is important for G-quadruplex-specific binding, suggesting modulation by specific structures of ncR-NAs [40].It is necessary for cis-repression of transcription induced by NTD of the Glu-rich domain, blocking its interaction with hsRPB7.EWS might function as a transcriptional regulator upon its binding with structure-specific RNAs [41].
EFPs may disturb gene expression by mimicking or interfering with CTD-Pol II normal function within the transcription initiation complex because EAD shares homology with CTD of Rpb1 [20].
EFPs may contribute to malignant transformation through disruption of RNA splicing, mediated by their binding proteins, such as YB-1 [45] and TASR proteins [46].Due to interaction with several RNA-processing proteins, including snRNP, U1C, and SF1 [8,44], EWS/FLI1 activity was linked to RNA transcription and splicing [45].The U1C expression modulates the EWS/FLI1 transactivation in vitro and in vivo via interaction with its NTD [8].RHA protein, a nuclear DNA/RNA helicase, regulating transcription and splicing, was found in a complex with EWS/FLI1 in ESFT cell lines, binding to EWS/FLI1 target gene promoters and enhancing its function as transcriptional cofactor [43].The complex formation between CBP/p300 and PolII requires RHA as modulator of transcription, inducing local changes in the chromatin structure [43,47].EWS/FLI1 specifically binds the RHA fragment AAs (630-1020), and the interruption of this interaction induces apoptosis in vivo and in vitro in ESFT cells [43].

The Common Role of EWS and EWS/FLI in Transcription
and Splicing of ESFT Cells.EWS is a bridge between basal transcriptional and splicing machinery of the gene expression [8,15,28,45], acting from the transcription initiation through to the delivery of the mature mRNA to the cytoplasm [16].EWS/FLI may have DNA-binding-independent dominant negative function, blocking the normal function of wild-type EWS expression [18].EWS is absent in RHA complex from HEK293 cell, while RHA possibly binds to wild-type EWS in ESFT [43].RHA may function differently in complexes with EWS versus EWS/FLI1, leading to oncogenic transformation in the presence of EWS/FLI1 [43].EWS/FLI1 and EWS share some protein partners such as BARD1 [48] but uniquely bind others such as YB1 [45].
The EAD of EWS/FLI1 is linked to mRNA splicing via U1C (snRNP) [8], indicating that both EWS/FLI1 and fulllength EWS can bind to U1C [49], while their direct interaction remains to be investigated.The functional consequences of heterodimerization between EWS/FLI1 and EWS on RNA splicing also have to be revealed [19,50].
All experimental data suggest a role of TET proteins in Pol II transcription and splicing, possibly coupling these processes.The participation of EWS and EFPs binding proteins in transcription and splicing could be described schematically by the model shown in Figure 2, as an attempt to link the transcription with the splicing.The partnerships  Evidently, the difference between EWS and EFPs binding to Pol II and TFIID is due to the nature of their CTDs.The EFP complex formation may be the result of structurally independent behavior of EAD and CTD, and their conformational changes.These interactions could be specific to tumor cell and may be undetectable in vitro as weak and unstable, allowing or disabling exact complex formations.Such binding element may be the YB-1 protein, connecting the C-terminus of EWS to TFIID and then recruiting them to Pol II, because, in ES cells (expressing endogenous EWS/FLI1), the linkage between YB-1 and Pol II via EWS (or TLS) was found to be defective [45].
The exact mechanisms of action and all interacting partners of EWS and EFPs, their role, and functional relationships in cancer pathways are not well understood.The function and connections of components of the model complexes, including dsDNA, mRNA, Pol II, EWS, EFPs, and other transcription and splicing factors, have to be revealed.The homoand heterodimerization function of EWS and EFPs in transcription and splicing, and also the direct interaction between EWS/FLI1 and U1 snRNP proteins have to be studied.

Conclusions
The proposed hypothetic model is trying to summarize the findings about functioning of EWS and EFPs and helps to understand the transcriptional and splicing mechanisms in humans.An important point is to find the functional consequences of EWS and EWS/FLI1 homo-and heterodimerization on RNA transcription and splicing.The discovery of new interacting partners of EWS and EFPs in vitro and in vivo as well as the inhibition of interaction, required for oncogenic activity, may help the development of uniquely effective, tumor-specific anticancer agents against Ewing's tumors.

Figure 2 :
Figure2: Schematic model of the link between transcription complex and spliceosome, reflecting interaction partners of EWS and EFPs.EWS is a bridge between components of basal transcriptional machinery and splicing apparatus of the general gene expression.EWS protein interacts via its RBD with nuclear ribonucleoproteins (hnRNPs) and RNA helicases.Shown are participants in transcription and splicing: EFP, hnRNPs, RPB7, EWS, YB-1, hnRNPs, RNA helicases, and other binding functional proteins.EWS could be divided into SYGQ-rich TAD and C-terminal RBD.This model shows to which regions of EWS and EFPs the interaction partners preferentially bind.Some interactions are depicted by arrows to better represent the unknown composition and possible flexibility of EWS and EFP containing complexes.