PRCC, the Commonest TFE3 Fusion Partner in Papillary Renal Carcinoma Is Associated with Pre-Mrna Splicing Factors

Oncogene (2001) 20, 178 ± 187 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors YM Skalsky*,1, PM Ajuh2, C Parker1, AI Lamond2, G Goodwin1 and CS Cooper1 1Institute of Cancer Research, Molecular Carcinogenesis Section, The Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK; 2Department of Biochemistry, The University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK In papillary renal cell carcinomas the TFE3 transcription shown to contain the PRCC ± TFE3, PSF ± TFE3 or factor becomes fused to the PSF and NonO pre-mRNA NonO ± TFE3 fusions, but not always reciprocal splicing factors and most commonly to a protein of fusions, they are the protein structures that believed unknown function designated PRCC. In this study we to be involved in carcinogenesis. have examined the ability of the resulting PRCC ± TFE3 TFE3 is a transcription factor that was originally and NonO ± TFE3 fusions to activate transcription from identi®ed by its ability to bind the mE3 site in the IgH the plasminogen activator inhibitor-1 (PAI-1) promoter. enhancer (Beckmann et al., 1990). The 576aa protein The results show that only fusion to PRCC enhanced contains a basic helix ± loop ± helix domain involved in transcriptional activation, indicating that the ability to DNA binding and two transcriptional activation enhance the level of transcription from endogenous TFE3 domains: an N-terminal acidic activation domain and promoters is not a consistent feature of TFE3 fusions. In a C-terminal proline rich activation domain (Artandi et investigations of the normal function of PRCC we al., 1995). A leucine zipper region is responsible for observed that PRCC expressed as a green ¯uorescent dimerization (Roman et al., 1992), homodimerization, fusion protein colocalizes within the nucleus with Sm pre- heterodimerization with the other family members, mRNA splicing factors. It was also found that such as TFEB, TFEC, the Microphthalmia protein endogenous PRCC is coimmunoprecipitated by anti- (Hemesath et al., 1994), and for binding with PU.1 of bodies that recognize a variety of pre-mRNA splicing the ETS protein family (Tian et al., 1999). A TFE3 factors including SC35, PRL1 and CDC5. Association target gene, which has been recently identi®ed, is the with the cellular splicing machinery is therefore, a plasminogen activator inhibitor-1 gene (PAI-1) (Hua et common feature of the proteins that become fused to al., 1998), which itself has been linked with oncogen- TFE3 in papillary renal cell carcinomas. Oncogene esis/metastasis (Bajou, 1998). TFE3 acts synergistically (2001) 20, 178±187. with SMAD3, which binds to an adjacent site in the PAI-1 promoter in the mediation of TGF,b induced Keywords: TFE3; PRCC; transcription activation; activation of PAI-1 (Hua et al., 1999). pre-mRNA splicing PSF is a ubiquitously expressed, essential pre-mRNA splicing factor (Patton et al., 1993). The full-length protein has 712 amino acids, which contain an N- Introduction terminal sequence rich in proline and glutamines and two consensus RNA binding domains. The PSF ± A recurrent translocation involving Xp11.2 is asso- TFE3 fusion protein contains almost all of PSF, fused ciated with papillary renal cell carcinomas. Several to the C-terminal portion of TFE3. The second variants of this translocation have been identi®ed partner, NonO (also called p54nrb) is the mammalian including t(X:1)(p11.2;q21), t(X;1)(p11.2;p34) and equivalent of Drosophila visual and courtship song inv(X)(p11.2;q12). In some cases alteration of Xp11.2 behaviour protein No-on-transient A (Yang et al., occur as the only cytogenetic abnormality (Meloni et 1993). NonO has signi®cant homology with PSF over a al., 1993; De Jong et al., 1986), suggesting that the 320aa region, encompassing the RNA binding domains genes involved in these translocations play an (Dong et al., 1993). This striking degree of homology important role in the process of cellular transforma- suggested that NonO was also a splicing factor and tion. Cloning of the breakpoints has revealed that the indeed it has been shown that its depletion from TFE3 gene at Xp11.2 is fused to either PRCC nuclear extract inhibits b-globin splicing in vitro chromosome 1q21, to PSF at 1p34 or to NonO at (Hallier et al., 1998). NonO may have a role in Xq12 (Sidhar et al., 1996; Weterman et al., 1996; Clark transcriptional activation (Basu et al., 1997) and was et al., 1997). Since all cases examined to date were also shown to bind PU.1 an ETS family protein that can alter the splicing activity of NonO in vitro (Hallier et al., 1996). PRCC codes for a protein of 491 amino acids with a *Correspondence: YM Skalsky Received 21 June 2000; revised 24 October 2000; accepted 24 proline, glycine and leucine rich N-terminal region. For October 2000 example, out of its 156 N-terminal amino acids 27% PRCC associates with splicing factors YM Skalsky et al 179 are proline. PRCC is not homologous to any known active than EGFP ± NonO ± TFE3. In fact EGFP ± protein and does not possess motifs indicative of NonO ± TFE3 failed to enhance activation when biochemical function. Recent studies have, however, compared to background level measured in cells shown that it contains domains capable of transcrip- transfected with PE2 reporter and GFP alone. Similar tional activation (Weterman et al., 2000). Analysis of relative levels of transactivation were observed when six cases of renal cell carcinoma with PRCC ± TFE3 assays were performed following TGF-b exposure. fusion genes has demonstrated two distinct breakpoints Cotransfection of SMAD3 further enhanced transcrip- in PRCC (Sidhar et al., 1996; Weterman et al., 1996). tional activation both before and after TGF-b In ®ve of the six cases, the fusion protein contained 156 treatment for EGFP ± PRCC ± TFE3 and EGFP ± amino acids of N-terminal PRCC fused to TFE3, while TFE3 but interestingly not for EGFP ± NonO ± TFE3 in the sixth case, 393 amino acids of N-terminal PRCC which again failed to enhance activation when were fused to TFE3. compared to background level measured in cells In this study we have undertaken a search for transfected with PE2 reporter, GFP alone and common features of the TFE3 fusions which could SMAD3. In these experiments EGFP ± PRCC ± TFE3 explain their oncogenic activity. Initial investigations showed twofold greater activity than EGFP ± TFE3, indicated that the ability to activate transcription from and ®vefold greater activity than EGFP ± NonO ± endogenous TFE3 promoters was not a common factor TFE3. All of these activities including the eects of of TFE3 fusions. Our studies were therefore extended TGF-b were dependent on the presence of an intact to examine the possibility that PRCC, in common with TFE3 binding site since mutations in the E-box NonO and PSF, may be a component of the pre- sequence from CACGTG to ACCGAC, which mRNA splicing machinery. abolishes TFE3 binding, ablated transcriptional activation in all experiments. The relative level of PAI-1 expression in papillary renal cell carcinomas cell lines PRCC ± TFE3(UOK Results 120), and NonO-TFE3(UOK 109) was measured by Northern blot analysis (Figure 1b) to con®rm the Activation of transcription results obtained by the TFE3 speci®c reporter assay It has recently been proposed, based on studies on the (Figure 1a). Neither UOK120 nor UOK109 harbour PRCC ± TFE3 fusion product that a key feature of normal copy of the TFE3 gene and therefore any TFE3 fusion partners is that they have the ability to TFE3 related changes on the transcription of PAI-1 enhance levels of transcription directed by TFE3 must be TFE3 fusion related. Our results showed that (Weterman et al., 2000). To assess whether this high levels of PAI-1 transcripts are present in UOK feature is common to other TFE3 fusion proteins we 120, but almost undetectable in UOK 109 as predicted compared the transcriptional activation properties of by the transactivation results. Equal levels of RNA PRCC ± TFE3 and NonO ± TFE3. All proteins were loading were demonstrated by the use of a GADPH expressed as enhanced green ¯uorescent protein probe. These results were further con®rmed by (EGFP) fusions so that the subcellular location of quantitative RT ± PCR analysis using oligo dT, the the protein could be examined in parallel studies. The random hexamer and PAI-1 speci®c primer for RT assay used relied upon the fact that the TFE3 followed by PCR using PAI-1 speci®c primers (data sequences present in PRCC ± TFE3 and NonO ± not shown). To further support the results presented in TFE3 fusions retain the TFE3 DNA binding domain. Figure 1a showing that the fusion NonO ± TFE3 failed To monitor the ability of each protein to activate to enhance activation of the PE2 reporter, a dual transcription we utilized a promoter sequence from luciferase reporter assay was used to verify whether the plasminogen activator inhibitor type I (PAI-1) Gal4 fusions of full length or the translocated region of gene fused upstream of the luciferase reporter gene NonO could activate transcription of a reporter (PE2 reporter). The PAI-1 promoter contains adjacent plasmid (Figure 1c). In these experiments NonO could TFE3 binding domain (E-box) and SMAD3 binding not activate transcription neither in HeLa nor in sites and therefore this construct can also be used to HepG2 cells which is in agreement with the results monitor interactions between TFE3 and SMAD3 in presented in Figure 1a. The fusion Gal4 ± TFE3 was response to signalling through the TGF-b/SMAD used as a positive control showing a marked transcrip- pathway.

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