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[Cancer Research 61, 3586-3590, May 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Oncogenic TLS/ERG and EWS/Fli-1 Fusion Proteins Inhibit RNA Splicing Mediated by YB-1 Protein1

Howard A. Chansky, Ming Hu, Dennis D. Hickstein and Liu Yang2

Department of Orthopedics, University of Washington School of Medicine, Seattle, Washington 98195 [H. A. C., M. H., L. Y.]; Medical Research Service, VA Puget Sound Health Care System, Seattle, Washington 98108 [H. A. C., M. H., L. Y.]; and Experimental Transplantation and Immunology, National Cancer Institute, Bethesda, Maryland 20892 [D. D. H.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The translocation liposarcoma protein TLS has recently been shown to function as an adapter molecule coupling gene transcription to RNA splicing. Here we demonstrate that YB-1, a protein known to play important roles in transcription and translation, interacts with the COOH-terminal domains of TLS and the structurally related Ewing’s sarcoma protein EWS. Through this interaction, YB-1 is recruited to RNA polymerase II and promotes splicing of E1A pre-mRNA to the 13S isoform. This splicing function of YB-1 is inhibited by exogenous TLS/ERG or EWS/Fli-1 fusion proteins, which bind to RNA polymerase II but fail to recruit the YB-1 protein. In Ewing’s sarcoma cells that express endogenous EWS/Fli-1, this linkage between YB-1 and RNA Pol II via EWS (or TLS) was found to be defective. Together, these results suggest that TLS and EWS fusion proteins may contribute to malignant transformation through disruption of RNA splicing mediated by TLS- and EWS-binding proteins such as YB-1.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The translocation liposarcoma protein TLS is a pro-oncoprotein whose COOH-terminal domain is replaced by C/EBP homologous protein in myxoid liposarcoma (1 , 2) and by an ETS transcription factor, ERG, in human myeloid leukemia (3) . We previously reported that TLS binds through its COOH-terminal domain to the SR3 family of splicing factors (4) . Because TLS binds to RNA Pol II through its NH2-terminal domain and interacts with SR proteins through its COOH-terminal domain, TLS may function as an adapter molecule linking gene transcription by RNA Pol II to RNA splicing by SR proteins. In recent studies we demonstrated that the TLS/ERG leukemia fusion protein not only inhibited alternative splicing promoted by transiently expressed SR proteins, but also interfered with constitutive splicing mediated by the basal splicing machinery (5) . To determine whether additional proteins might also function as splicing factors that use TLS as an adapter, we carried out experiments involving YB-1 protein, which was identified through yeast two-hybrid screen.

YB-1 is a multifunctional protein that shuttles between the cytoplasm and the nucleus (6) . In the cytoplasm, YB-1 binds to mRNA and regulates mRNA stability and translation efficiency. In the nucleus, YB-1 binds to specific promoter sequences and regulates transcription of diverse target genes, including multidrug resistance-1, a gene that contributes to resistance to chemotherapy in human breast cancer and osteosarcoma (7 , 8) . Although nuclear YB-1 has been identified at sites of active gene transcription, it is not known whether YB-1 plays a role in RNA splicing (6) .

In this report we describe the identification of YB-1 as a protein that interacts with TLS as well as with the structurally related Ewing’s sarcoma protein EWS. We also demonstrate that YB-1 associates specifically with the hyperphosphorylated form of RNA Pol II and influences splice site selection of E1A pre-mRNA transcripts and that this splicing function of YB-1 is inhibited by oncogenic TLS and EWS fusion proteins.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Plasmid Construction.
The TLS bait plasmid used in yeast two-hybrid screen was generated by inserting the human TLS sequence into the pBTM116 vector. cDNA for human YB-1 was cloned into the EcoRI-XbaI sites of pCS2-MT vector to generate a YB-1 expression plasmid with the Myc epitope tagged at the NH2 terminus. The construction of TLS and EWS expression plasmids has been described previously (5 , 9) . For convenient detection, the full-length YB-1 cDNA in pSG5-YB-1 (10) was tagged at the COOH terminus with the influenza HA epitope to generate pSG5-HA-YB-1. The adenovirus E1A splicing reporter construct pCS3-MT-E1A was obtained from Dr. Moreau-Gachelin (Institut Curie, Paris, France) (11) , and hnRNP A1 cDNA was cloned into the EcoRI-XbaI sites of the pCR3 vector (Invitrogen).

Two-Hybrid Screen.
The yeast two-hybrid library was constructed with mRNAs from the growth factor-dependent hematopoietic EML cells (12) . The two-hybrid screen was performed as described previously (4) . In brief, L40 yeast harboring the bait plasmid pBTM-TLS were transformed with 300 µg of the EML cDNA library in which the cDNAs were fused to the transactivation domain of VP16 protein. Interaction of the TLS bait with its target proteins transactivated both the HIS3 and the LacZ genes in the transformants. Positive clones were thus identified through X-gal assay and growth in selective medium. The L40 clones were then cured of the bait plasmid and mated with AMR70 yeast containing either pBTM-TLS or various control plasmids to confirm the specificity of protein-protein interactions.

Immunoprecipitation and Western Blot Analysis.
For protein expression in COS-7 cells, 10 µg of pSG5-Flag-expression plasmid and 10 µg of pCS2-MT-YB-1 were introduced into 3 x 106 cells by electroporation. For protein expression in Ewing’s sarcoma cell line SK-N-MC, 5 µg of pSG5-Flag-expression plasmid and 5 µg of pCS2-MT-YB-1 were mixed with 60 µl of lipofection reagent DOTAP (Roche Molecular Biochemicals). The DNA-DOTAP mixture was added to 65% confluent SK-N-MC cells in a 100-mm dish according to the manufacturer’s instructions. Forty-eight h after electroporation or DOTAP transfection, the cells were lysed with 0.6 ml of lysis buffer A [10 mM Tris-HCl (pH 7.4), 2.5 mM MgCl2, 100 mM NaCl, 0.5% Triton X-100]. Eight µl of mouse monoclonal 9E10 anti-Myc antibody (Sigma Chemical Co.) or 10 µl of mouse monoclonal 8WG16 anti-Pol II antibody (Research Diagnostics, Inc) were first incubated with 30 µl of protein A/G agarose (Santa Cruz Biotechnology) for 50 min at 4°C in 0.3 ml of buffer A. The antibody-protein A/G-agarose complex was then incubated with 0.2 ml of lysate for 20 min at 4°C with gentle rocking. After the beads were washed four times with RIPA buffer, 50 µl of SDS-PAGE sample buffer were added. The samples were heated at 100°C for 5 min, 10 µl of the sample were separated by SDS-PAGE, and the proteins were detected with the mouse monoclonal M2 anti-Flag antibody (Sigma Chemical Co.), the mouse monoclonal H14 anti-Pol II antibody (Research Diagnostics, Inc), or the rabbit polyclonal C-21 anti-Pol II antibody (Santa Cruz Biotechnology). Protein bands were visualized using the ECL Western Blotting Analysis System (Amersham).

In Vivo Splicing Assay.
For in vivo splicing of E1A pre-mRNA, 2 µg of pCS3-MT-E1A, 4 µg of pSG5-HA-YB-1, and 4 µg of pSG5-Flag-construct were mixed with 60 µl of DOTAP. The DNA-DOTAP mixture was added to two 60-mm dishes with 65% confluent NIH/3T3 cells in 4 ml of DMEM containing 1% fetal bovine serum. After incubation for 40 h with the DNA-DOTAP mixture, the cells from one dish were lysed with 0.25 ml of RIPA buffer for Western blotting with the M2 anti-Flag antibody and the F-7 anti-HA antibody (Santa Cruz Biotechnology). Cells from the other dish were lysed for RNA isolation with an RNeasy column (Qiagen).

For the RNase protection assay, 10 µl of total RNA were hybridized to 1.5 x 106 cpm of 32P-labeled RNA probe antisense to the E1A genomic sequence (covering bases 499-1316 of the E1A gene) or to 1 x 106 cpm of 32P-labeled control antisense RNA probe covering the entire coding region of hnRNP A1. After overnight hybridization, excess RNA probe was digested with a mixture of RNase A + T1 supplied with the RNase Protection Assay System (PharMingen). The protected antisense E1A or hnRNP A1 fragments were isolated according to the manufacturer’s instructions, denatured, and separated on a 6% QuickPoint precast denaturing gel (Novex).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
YB-1 Interacts with TLS and EWS Pro-Oncoproteins.
To identify potential splicing factors that may interact with the COOH-terminal domain of TLS, we carried out screening of a yeast two-hybrid library from a mouse hematopoietic cell line. In addition to SR splicing factors SC35, TASR-1, and TASR-2 that we previously identified to be partners of TLS, the transcription and translation factor YB-1 was the most frequently encountered protein that interacted with the TLS bait. In different rounds of screening, more than seven independent YB-1 clones were isolated, and sequencing of their inserts revealed that these clones all corresponded to the COOH-terminal tail domain of YB-1 (Fig. 1)Citation . On the basis of sequence alignment, the COOH-terminal domain of YB-1 between amino acid 170 and 258 is responsible for interaction with the COOH-terminal domain of TLS.



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  		Fig. 1. Schematic of YB-1 clones that interact with TLS. Full-length human YB-1 protein is shown with cold shock domain and tail domain indicated. Seven independent YB-1 clones were found to interact with the TLS bait in the yeast two-hybrid screen. Each YB-1 clone is shown at left with the corresponding amino acid sequence in parentheses and the corresponding region aligned at right.

 
Our finding that TLS interacts with YB-1 in the yeast two-hybrid system suggested that these two proteins might also associate with each other in mammalian cells. To confirm their intracellular association, plasmids expressing Flag-tagged wild-type TLS, ERG, the TLS/ERG leukemia fusion protein, and Myc-tagged YB-1 were cotransfected into COS-7 cells, and lysates from the cotransfected cells (Fig. 2ACitation , Lanes1–3) were used for immunoprecipitation. Flag-TLS formed a protein complex with Myc-YB-1, and the complex was immunoprecipitated with a mouse monoclonal anti-Myc antibody (Fig. 2ACitation , Lane 4). In contrast, Flag-TLS/ERG and Flag-ERG failed to associate with Myc-YB-1 (Fig. 2ACitation , Lanes 5 and 6).



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  		Fig. 2. Interactions of YB-1 with TLS and EWS in COS-7 cells. A, pCS2-MT-YB-1 plasmid expressing Myc-tagged YB-1 was cotransfected into COS-7 cells with plasmid expressing Flag-tagged TLS, TLS/ERG, or ERG (Lanes 1–3). The lysates were immunoprecipitated (IP) with the 9E10 anti-Myc antibody (Lanes 4–6); the immunoprecipitates were separated by SDS-PAGE on a 10% gel and blotted with the M2 anti-Flag antibody (Blot: anti-Flag) or the 9E10 anti-Myc antibody (Blot: anti-Myc). B, COS-7 cells were transfected with pCS2-MT-YB-1 plus plasmid expressing Flag-EWS, EWS/Fli-1, or Fli-1 (Lanes 1–3). The lysates were immunoprecipitated with the anti-Myc antibody (Lanes 4–6) then blotted with the anti-Flag antibody.

 
The Ewing’s sarcoma protein EWS shares sequence homology with translocation liposarcoma protein TLS (13) . Ewing’s sarcoma and several other tumors are characterized by chromosomal translocations involving EWS. Similar to TLS fusion proteins, the COOH-terminal domain of EWS is replaced by the corresponding fusion partner, whereas the NH2-terminal domain of EWS is retained, implying that both TLS and EWS fusion proteins transform cells through a similar mechanism (14) . To investigate whether YB-1 also interacts with the COOH-terminal domain of EWS, Flag-tagged wild-type EWS, Fli-1, and the EWS/Fli-1 sarcoma fusion protein were coexpressed in COS-7 cells along with Myc-tagged YB-1 (Fig. 2BCitation , Lanes 1–3). EWS formed a protein complex with YB-1 (Fig. 2BCitation , Lane 4), whereas EWS/FLI-1 and Fli-1 were absent from the immunoprecipitates. On the basis of the results from our yeast two-hybrid screen and from the coimmunoprecipitation experiments, the COOH-terminal domains of TLS and EWS were shown to interact with the tail domain of YB-1 in mammalian cells.

YB-1 Is Recruited to Hyperphosphorylated RNA Pol II.
Although gene transcription and RNA splicing can be carried out separately in vitro, experimental evidence now indicates that these two processes are tightly coupled in vivo through the COOH-terminal YSPTSPS heptapeptide repeats of the largest RNA Pol II subunit (15) . Prior to initiation of transcription, the COOH terminus of this largest subunit of RNA Pol II is hypophosphorylated (Pol IIa form). After initiation of transcription, these heptad repeats become phosphorylated, and splicing factors are recruited to the COOH-terminal domain of hyperphosphorylated RNA polymerase (Pol IIo form). The NH2-terminal domains of both TLS and EWS have been reported to be responsible for interaction with RNA Pol II (5 , 9) ; thus both TLS and EWS, along with their fusion proteins, were found in RNA Pol II immunoprecipitates (Fig. 3ACitation , Lanes 7, 8, 10, 11). ERG and Fli-1 were absent from the immunocomplexes, indicating that they do not interact with RNA Pol II (Fig. 3ACitation , Lanes 9 and 12). The 8WG16 anti-Pol II antibody immunoprecipitated both hyper- and hypophosphorylated forms of RNA Pol II (9) ; therefore, the phosphorylation status of RNA Pol II that associated with TLS and EWS could not be determined through immunoprecipitation with 8WG16. To examine whether YB-1 coimmunoprecipitated with hyperphosphorylated RNA Pol II, COS-7 lysates coexpressing Flag-TLS, EWS, and Myc-YB-1 were immunoprecipitated with an anti-Myc antibody. The immunoprecipitates were washed under mild detergent conditions (9) , and then blotted with H14 and C-21 antibodies that specifically recognize RNA Pol IIo or Pol IIa, respectively. The results indicated that Myc-YB-1 is associated with hyperphosphorylated RNA Pol IIo as well as with Flag-TLS and Flag-EWS (Fig. 3BCitation , Lanes 2 and 5).



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  		Fig. 3. YB-1 association with hyperphosphorylated RNA Pol II. A, COS-7 lysates expressing Flag-tagged proteins (Lanes 1–6) were immunoprecipitated (IP) with 8WG16 anti Pol-II antibody (Lanes 7–12). The immunoprecipitates were separated and blotted with the anti-Flag antibody. B, COS-7 cells were cotransfected with Myc-YB-1 plus Flag-TLS (Lanes 1–3) or Myc-YB-1 plus Flag-EWS (Lanes 4–6). The lysates (Lanes 1 and 4) were immunoprecipitated with the 9E10 anti-Myc antibody (Lanes 2 and 4) or a control mouse IgG (Lanes 3 and 6). The samples were separated on a 6% gel and blotted with the H14 antibody, which recognizes RNA Pol IIo (Blot: H14), the C-21 antibody, which recognizes RNA Pol IIa (Blot: C-21), or the M2 anti-Flag (Blot: anti-Flag).

 
The experiments described above cannot discriminate between the possibilities that the YB-1-Pol II complex requires docking by TLS and EWS or that there are two pools of YB-1 in the cells: one pool associates with TLS or EWS, whereas the other YB-1 pool associates with RNA Pol II on its own. To address this concern, we carried out additional transfection experiments to express Flag-EWS and Myc-YB-1 in the Ewing’s sarcoma cell line SK-N-MC. If YB-1 associates with RNA Pol II without docking through EWS or TLS, then YB-1 should be coimmunoprecipitated with Pol II regardless of the presence of endogenous EWS/Fli-1. Conversely, the association of YB-1 with Pol II may be affected by endogenous EWS/Fli-1 in Ewing’s sarcoma cells if docking via EWS or TLS is required. After transfection, both Myc-YB-1 and Flag-EWS were expressed in SK-N-MC cells (Fig. 4Citation , middle and bottom panels, Lane 1). Immunoprecipitation with the anti-Myc antibody concentrated the Myc-YB-1 from the lysate, but Flag-EWS protein was absent from the immunoprecipitates either with the anti-Myc antibody or with a control IgG (Fig. 4Citation , Lanes 2 and 3). Similar results were also obtained when SK-N-MC cells coexpressing Myc-YB-1 and Flag-TLS were analyzed (Fig. 4Citation , Lanes 4–6). Unlike COS-7 cells, from which Myc-YB-1 was coimmunoprecipitated with Pol II and Flag-TLS (or EWS), in SK-N-MC Ewing’s sarcoma cells RNA Pol II was not detected from the anti-Myc immunoprecipitates (Fig. 4Citation , top panel, Lanes 2 and 5). These results suggest that YB-1 on its own does not interact with RNA Pol II and that the association of YB-1 with Pol II via wild-type TLS and EWS proteins is defective in SK-N-MC Ewing’s sarcoma cells.



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  		Fig. 4. Disassociation of YB-1 from EWS, TLS, and RNA Pol II in Ewing’s sarcoma cells. SK-N-MC lysates expressing Myc-YB-1 plus Flag-EWS (Lane 1) or Myc-YB-1 plus Flag-TLS (Lanes 4) were immunoprecipitated (IP) with the 9E10 anti-Myc antibody (Lanes 2 and 4) or a control mouse IgG (Lanes 3 and 6). The samples were separated on a 6% gel and blotted with the H14 anti-Pol II antibody (Blot: H14), the M2 anti-Flag antibody (Blot: anti-Flag), or the 9E10 anti-Myc antibody (Blot: anti-Myc).

 
YB-1-mediated RNA Splicing Is Inhibited by TLS and EWS Fusion Proteins.
In SK-N-MC Ewing’s sarcoma cells, YB-1 is not able to promote adenovirus E1A pre-mRNA splicing (data not shown). Because endogenous EWS/Fli-1 fusion protein might disrupt YB-1 association with hyperphosphorylated RNA Pol IIo in these Ewing’s sarcoma cells, we investigated the splicing activity of YB-1 in NIH/3T3 cells, which are not known to harbor any TLS or EWS translocation. The alternative splicing of E1A pre-mRNA generates five different splicing products, designated 13S, 12S, 11S, 10S, and 9S mRNA (Fig. 5A)Citation , and each E1A splicing product can be identified according to the distinct sizes of the protected RNA fragments in the RNase protection assay. In the absence of exogenous YB-1 protein expression, the major splicing products of E1A pre-mRNA in NIH/3T3 cells were the 13S, 12S, and 9S isoforms (Fig. 5BCitation , Lane 1). YB-1 overexpression prominently enhanced splicing to the 13S isoform (Fig. 5BCitation , Lane 2), and this observation suggests that YB-1 can influence splice site selection in the manner of a typical splicing factor. Coexpression of wild-type TLS and EWS did not significantly alter E1A pre-mRNA splicing mediated by YB-1 (Fig. 5BCitation , Lanes 3 and 5), possibly because of high levels of endogenous TLS and EWS in NIH/3T3 cells. Interestingly, coexpression of either TLS/ERG leukemia fusion protein or EWS/Fli-1 sarcoma fusion protein leads to inhibition of the 13S isoform promoted by YB-1 (Fig. 5BCitation , Lanes 4 and 6). The TLS-associated splicing factor TASR-1 is known to promote splicing of E1A pre-mRNA to the 11S, 10S, and 9S isoforms (4) ; therefore, it was transfected into NIH/3T3 cells as a control to differentiate the E1A splicing products from background bands (Fig. 5BCitation , Lane C). To ensure equivalent RNA input, the unrelated hnRNP A1 message was also analyzed by RNase protection assay, and similar levels were found in all samples (Fig. 5BCitation , panel labeled Probe: anti-sense hnRNP A1). Through Western blotting, the levels of Flag-tagged TLS, TLS/ERG, EWS, EWS/Fli-1, and HA-tagged YB-1 proteins were found to be comparable, demonstrating that the observed inhibition of E1A splicing was not attributable to variation in transfection or YB-1 expression (Fig. 5BCitation , panels labeled IP: anti-flag/Blot: anti-Flag and Blot: anti-HA). On the basis of these results, we conclude that TLS/ERG and EWS/Fli-1 fusion proteins most likely exert their influence by blocking the recruitment of YB-1 to RNA Pol II splicing machinery, leading to inhibition of splice site selection favored by YB-1.



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  		Fig. 5. Inhibition of YB-1-mediated RNA splicing by TLS/ERG and EWS/Fli-1 fusion proteins. A, diagram of individual E1A pre-mRNA splicing products. Numbers indicate the individual exons, and dashed lines represent spliced sequences. B, effects of TLS and EWS fusion proteins on YB-1-mediated E1A pre-mRNA splicing were analyzed in NIH/3T3 cells by RNase protection assay (Lanes 1–6 and C). DNA combinations for all samples are indicated at the top of the panel. Positions of RNA markers are labeled at the left, and protected E1A RNA fragments are shown on the right with exons designated by numerals in boxes. The protected antisense hnRNP A1 fragment is shown in the panel labeled Probe: anti-sense hnRNP A1, immunoprecipitated Flag-proteins are shown in the panel labeled IP: anti-Flag/Blot: anti-Flag, and the HA-tagged YB-1 is shown in the panel labeled Blot: anti-HA. The position of the anti-Flag antibody is indicated by an *.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
YB-1 has been well documented as a transcription factor and as a regulator of translation. Our demonstration that the tail domain of YB-1 interacts with the COOH-terminal domains of TLS and EWS, which in turn dock YB-1 to RNA Pol II to participate in pre-mRNA splicing, expands the known functions of YB-1 protein. It is likely that YB-1 plays an important role in the coordinated control of transcription, splicing, and translation in mammalian cells.

TLS, EWS, and the TATA-binding protein-associated factor TAFII68 (Ref. 16 ; collectively called TET proteins) belong to a family of RNA-binding proteins that share a high degree of sequence homology. In a variety of human cancers, TET proteins are fused to diverse nuclear partners such as ATF-1, C/EBP homologous protein, ERG, ETV1, E1A-F, FEV, Fli-1, TEC1, and WT1 (17) . The common feature among different TET fusion proteins is the retention of their NH2-terminal domains and the replacement of their COOH-terminal domains by the fusion partners. The NH2-terminal domains of TLS, EWS, and TAFII68 have been shown to be functionally interchangeable in transactivation and transformation assays; therefore, TET fusion proteins are thought to cause cellular transformation as chimeric transcription factors (14) . However, deletion studies of EWS/Fli-1 have indicated that the NH2-terminal subdomain required for transformation differs from the subdomain required for transactivation (18) , and site-directed mutagenesis studies have shown that an EWS/Fli-1 mutant lacking DNA-binding ability still possesses the ability to transform cells (19) . In view of these discrepancies, it is possible that disruption of other cellular processes, such as RNA splicing, may contribute to cellular transformation by TET fusion proteins.

The control of RNA splicing represents an important step in eukaryotic gene expression, and aberrant RNA splicing products are frequently found in cancer cells, including those of Ewing’s sarcoma (20) . Our finding that wild-type TET proteins function as adapter molecules coupling gene transcription to RNA splicing provides a potential alternative mechanism for oncogenic transformation by TET fusion proteins. TLS, EWS, and TAFII68 may normally bind to RNA Pol II and recruit various splicing factors to the sites of active transcription. In cells harboring a TET fusion protein, the NH2-terminal domain of the TET fusion protein still binds to RNA Pol II, but the COOH-terminal domain of the fusion protein fails to recruit splicing factors because of replacement by the fusion partner. In addition to NIH/3T3 cells, TLS/ERG and EWS/Fli-1 also interfered with YB-1-mediated splicing in COS-7 and HeLa cells despite the presence of endogenous TLS and EWS (data not shown), TET fusion proteins therefore appear to abrogate splicing functions of the wild-type EWS and TLS proteins in a dominant-negative manner. This notion is further supported by our finding that the linkage between YB-1 and RNA Pol II via EWS (or TLS) is defective in SK-N-MC Ewing’s sarcoma cells.

Disruption of the coordinated processes of transcription and splicing would be expected to result in degradation of the unprocessed pre-RNA or generation of aberrant splicing products. Supporting this concept is the fact that a majority of TET-interacting proteins identified to date are splicing factors such as SF1, Spi-1, YB-1, RNPs A1 and U1C, and SR proteins SC35, TASR-1, and TASR-2 (see Ref. 9 ). Alternative splicing of important transcripts such as CD44, a molecule associated with tumor cell growth and metastasis, is also affected by the TLS/ERG leukemia fusion protein (5) . In this study we have shown that YB-1-mediated splicing of E1A reporter transcripts is inhibited by TLS/ERG and EWS/Fli-1. In future experiments we will focus on identifying critical endogenous splicing targets disrupted by TET fusion proteins.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by funds from the Department of Orthopedics and Sports Medicine at the University of Washington and by a grant from the National Leukemia Research Association (to L. Y.). Back

2 To whom requests for reprints should be addressed, at Department of Orthopedics, GMR-151, University of Washington School of Medicine, 1660 S. Columbian Way, Seattle, WA 98108. Phone: (206) 277-6913; Fax: (206) 768-5261; E-mail: lyang{at}u.washington.edu Back

3 The abbreviations used are: SR, serine-arginine; Pol, polymerase; HA, hemagglutinin; hnRNP, heterogeneous nuclear ribonucleoprotein. Back

Received 10/12/00. Accepted 3/13/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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