The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb.

The protooncogene for Spi-1/PU.1 is an Ets-related transcription factor overexpressed during Friend erythroleukemia. The molecular basis by which Spi-1/PU.1 is involved in the erythroleukemic process remains to be elucidated. By using an immobilized protein binding assay, we have identified a 55-kDa protein as a putative partner of Spi-1/PU.1 protein. Microsequence analysis revealed that this 55-kDa protein was p54nrb (nuclear RNA-binding protein, 54 kDa) a RNA-binding protein highly similar to the splicing factor PSF (polypyrimidine tract-binding protein-associated splicing factor). In this paper, we show that Spi-1/PU.1 impedes the binding of p54nrb to RNA and alters the splicing process in vitro. Moreover, we present evidence that the transcriptional factor Spi-1/PU.1, unlike other Ets proteins, is able to bind RNA. Altogether, these results raise the intriguing possibility that the functional interference observed between Spi-1/PU.1 and RNA-binding proteins might represent a novel mechanism in malignant erythropoiesis.

In the Friend spleen focus forming virus-induced erythroleukemia, the insertional mutagenesis of the spi-1 gene appears to be related to the emergence of a clonal population of tumorigenic erythroid cells arrested in their differentiation at the proerythroblast stage. Such activation of spi-1 results in an overexpression of the normal Spi-1/PU.1 protein in the Friend tumor cells (1). spi-1 encodes the PU.1 protein, a member of the Ets family of transcription factors (2). Its DNA binding domain targets specific sequences around a central core 5Ј-GGAA-3Ј in transcriptional promoters and enhancers of some myelomonocytic, mastocytic, and B lymphoid genes (3). Spi-1/PU.1 contains also an amino-terminal transactivator domain and a central PEST region that could be involved in interactions with proteins like the retinoblastoma protein (4), the transcription factor TFIID (4), and the factor NF-EM5 (5) or Pip (6). The down-regulation of spi-1 during the chemically induced differentiation of the Friend tumor cells (7) and the severe anemia developed by the Spi-1/PU.1 transgenic mice (8) suggest that spi-1 plays a role in the transformation of the proerythroblast by blocking its differentiation. The oncogenic potential of Spi-1/PU.1 may result from targeting of inappropriate regulatory elements of some erythroid genes and/or an abnormal association with erythroid partners. In order to determine whether Spi-1/PU.1 interacts specifically with nuclear proteins from Friend cells, a glutathione S-transferase-Spi-1/PU.1 fusion protein was used as affinity chromatographic reagent. We report here that the RNA-binding protein p54 nrb (9) was identified by this procedure as a putative partner of Spi-1/PU.1. In addition, this study reveals the ability of Spi-1/PU.1 to bind RNA and to interfere in vitro with splicing process. This novel property of Spi-1/PU.1, characterized as a DNA-binding transcriptional regulator, provides new insights into the molecular mechanism involved in malignant hematopoiesis.
Electrophoretic Mobility Shift Assay-In vitro translated Spi-1/PU.1 protein was incubated with 32 P-5Ј-end-labeled E3Ј DNA probe (5) in 20 mM HEPES (pH 7.5), 50 mM KCl, 2 mM MgCl 2 , 10% glycerol with 1.5 g of poly(dI-dC)/20-l reaction. Reactions were incubated at 25°C for 15 min. For competitive experiments, the E3Јprobe was incubated with Spi-1/PU.1 before addition of 250 ng of homoribopolymers (Pharmacia Biotech Inc.). RNA were transcribed in vitro using [␣-32 P]UTP and SP6 * This work was supported by the Ligue Nationale Française contre le Cancer and the Association pour la Recherche sur le Cancer. 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.
‡ Supported by a fellowship from the Société Française d'Hématologie.
polymerase following the recommendations of the supplier (Promega). The gel mobility shift assay was carried out with 32 P-labeled RNA polypyrimidine tract of the ␤-tropomyosin intron probe (13) and GST fusion proteins. The reaction mixture for binding was 20 mM HEPES (pH 7.5), 50 mM KCl, 10% glycerol, and 1 g of yeast transfer RNA.
Northwestern Blot-Northwestern blotting was carried out according to Crozat et al.(14).
In Vitro Splicing-A human ␤-globin minigene from exon 1 to exon 2 was transcribed and 32 P-labeled in vitro from the plasmid pSP64 (16). Splicing reactions (25 l) were carried out with 10 l of HeLa nuclear extracts in a buffer containing 3.2 mM MgCl 2 , 40 mM KCl, 0.5 mM ATP, 20 mM creatine phosphate, 1% (w/v) polyvinyl alcohol, and 4 ng of 32 P-labeled ␤-globin pre-mRNA. After 3 h of incubation at 30°C the RNA was purified and analyzed on 6% polyacrylamide gel in 8 M urea, as described elsewhere (16). GST and GST-fusion proteins were incubated for 10 min with HeLa cell nuclear extracts (11) before addition of 32 P-labeled ␤-globin mRNA. For preclearing assay, 50 l of HeLa cell nuclear extracts were incubated for 1 h at 4°C with 20 l of Sepharose-GST or Sepharose-GST-Spi-1 proteins equilibrated in buffer D (11). After centrifugation, 15 l of precleared extracts were used in the splicing assay.

RESULTS AND DISCUSSION
A glutathione S-transferase fusion protein containing the murine Spi-1/PU.1 protein (GST-Spi-1) bound to glutathione-Sepharose was used as affinity chromatographic reagent to search for putative partners of Spi-1/PU.1. GST-Spi-1 was incubated with nuclear extracts from the murine Friend erythroleukemia cell line 745A. The proteins recovered on GST-Spi-1-glutathione-Sepharose beads were analyzed by SDS-PAGE. One protein, with apparent molecular mass around 55 kDa, was retained specifically by the GST-Spi-1 fusion protein (data not shown). This 55-kDa protein was purified on SDS-polyacrylamide gel. 2 A 15-amino acid internal peptide was subjected to amino acid sequence determination. A data base search revealed that this sequence (LEMEMEAARHEHQVM) perfectly matched to the nuclear RNA-binding protein p54 nrb (54 kDa) (9). p54 nrb is highly related to the human splicing factor PSF (PTB-associated splicing factor) (17) in a 320-amino acid region containing a RNA binding domain with two RNA recognition motifs (RRM/RBD/RNP-CS) (Fig. 1A).
Spi-1/PU.1 and its related protein Spi-B (18), are the most phylogenetically divergent members of the Ets family. Their Ets domains are 70% homologous and present only 35-40% sequence identity with that of Fli-1 (19) and Ets-2 (20). The specificity of binding of p54 nrb to Spi-1/PU.1 was approached by investigating whether other Ets proteins: Fli-1, Ets-2, and Spi-B, were able to interact with p54 nrb . GST-p54 nrb fusion protein was incubated in the presence of 35 S-labeled Spi-1/ PU.1, Spi-B, Ets-2, and Fli-1 proteins translated in reticulocyte lysates (Fig. 1B). Only Spi-1/PU.1 and Spi-B bound GST-p54 nrb , revealing that in vitro association of p54 nrb with Spi proteins is not a general property of the Ets proteins.
Spi-1/PU.1 contains three domains: the transactivation domain (amino acids 1-111), the PEST domain (amino acids 111-158), and the DNA-binding domain (DBD) including the Ets motif (amino acids 158 -266). The Spi-1/PU.1 domain (Fig.  1A) involved in the association of Spi-1/PU.1 with p54 nrb was mapped by testing interactions of 35 S-labeled in vitro translated p54 nrb with various deleted forms of GST-Spi-1 (Fig. 1A). Data in Fig. 1C showed that only the entire Spi-1/PU.1 (GST-Spi-1) and its DNA-binding domain (GST-DBD-Spi-1) interacted with p54 nrb protein. The same results were obtained for Spi-B (data not shown). In contrast, the DBD of Fli-1 fused to GST did not interact with p54 nrb . In these experiments, we ascertained that the DNA binding domains of Spi-1/PU.1 and Fli-1 fused to GST were able to bind their respective responsive element in band shift assay (data not shown). p54 nrb , like PSF (17), contains a central RNA binding domain with two RNA recognition motifs (RRM). Different truncated GST-p54 nrb fusion proteins (Fig. 1A) were tested for their abilities to bind in vitro 35 S-translated Spi-1/PU.1 protein. Interactions occurred only between the entire GST-p54 nrb protein or the GST-RBD-p54 nrb , suggesting that p54 nrb bound Spi-1/PU.1 by its RNA binding domain. The RNA binding activities of GST-p54 nrb and GST-RBD-p54 nrb were controlled on a Northwestern blot probed with 32 P-labeled poly(A) ϩ mRNAs from 745A cells (Fig.  4A). Altogether, these data suggested that the interaction of 2 M. Hallier, A. Tavitian, and F. Moreau-Gachelin, manuscript in preparation. Then, we searched whether p54 nrb and Spi-1/PU.1 could be coimmunoprecipitated when coexpressed in COS cells (Fig. 2). Nuclear extracts from transfected COS cells were immunoprecipitated under low stringency conditions with the antibody against 9E10 Myc epitope (12) used to tag p54 nrb (tag-Myc-p54 nrb ). The presence of Spi-1/PU.1 in Myc immunoprecipitates was assessed by immunoblotting with an antibody against Spi-1/PU.1 (7) and was detected only in COS cells transfected with both Spi-1/PU.1 and p54 nrb expression vectors. This provided evidence that Spi-1/PU.1 was associated with p54 nrb in vivo.
The interaction between the DBD of Spi-1/PU.1 and the RBD of p54 nrb suggested that it could alter the function of each partner. p54 nrb altered neither the binding of Spi-1/PU.1 on various DNA-responsive elements nor the transcriptional activity of Spi-1/PU.1 in CAT assay (data not shown). We sought to discover whether Spi-1/PU.1 might change the behavior of the RNA-binding protein p54 nrb . The pyrimidine-rich sequence of the branchpoint/polypyrimidine tract RNA, which is part of most 3Ј splice sites in mammalian introns, is targeted by PSF (17). Since p54 nrb presents 70% identity with PSF in its RRM, we tested whether p54 nrb could bind such RNA sequence. We observed (Fig. 3) that, like PSF, p54 nrb binds the pyrimidinerich sequence of the 3Ј splice site in the intron of the ␤-tropomyosin pre-mRNA (13). Thus, this RNA sequence was used as probe in EMSA (Fig. 3). Increasing amounts of GST-Spi-1 mixed with an equal input of GST-p54 nrb reduced the formation of the GST-p54 nrb -RNA complex in a dose-dependent manner, revealing that Spi-1/PU.1 impedes the binding of p54 nrb to RNA.
The binding of Spi-1/PU.1 to the RNA-binding protein p54 nrb prompted us to check whether Spi-1/PU.1 binds RNA. Various deletion mutants of Spi-1/PU.1 and p54 nrb fused to GST were analyzed by Northwestern blot using the labeled poly(A) ϩ mRNAs from 745A cell line as a probe. Fig. 4A revealed that Spi-1/PU.1, by its DNA binding domain, was able to interact with RNAs as the RNA-binding domain of p54 nrb . To further investigate the interaction of Spi-1/PU.1 with RNA, we tested its ability to bind homoribonucleotide polymers fixed to agarose beads. Spi-1/PU.1 (and Spi-B not shown) bound preferentially the homoribopoly(G) (Fig. 4B). This affinity of Spi-1/PU.1 for poly(G) appeared functionally relevant since in EMSA performed with the Spi-1/PU.1 DNA-responsive element E3Ј (5), an excess of poly(G) competed the binding of Spi-1/PU.1 to DNA sequence (Fig. 4C). Elsewhere, in competitive experiments (Fig. 3), p54 nrb exhibited affinity for poly(G) and poly(U) revealing that Spi-1/PU.1 and p54 nrb display a similar ability to bind poly(G). In agreement with the absence of in vitro-interaction between p54 nrb and Fli-1 or Ets-2, we observed that the DNA binding activity of Fli-1 fused to GST was not affected by an excess of homoribopolymers (data not shown) and that Fli-1 and Ets-2 did not bind any homoribopolymers (Fig. 4B). These data brought a first evidence that the transcription factor Spi-1/PU.1 was able to bind RNA.
Due to the extensive homology between p54 nrb and PSF, a factor involved in RNA splicing, we asked whether Spi-1/PU.1 might interfere with the splicing process. In vitro splicing reactions were performed with HeLa cell nuclear extracts and a pre-mRNA transcribed from a minigene containing human ␤-globin exons 1 and 2. The addition of GST-Spi-1/PU.1 inhibited the formation of the spliced transcript, whereas addition of another Ets protein, like GST-Fli-1 protein, did not (Fig. 5). This suggests that alteration of splicing process did not result from an excess of an Ets protein in splicing extracts. Moreover, the DNA-binding domain of Spi-1/PU.1 that contains the RNA binding activity appeared sufficient to inhibit the formation of the spliced transcript (Fig. 5). Noteworthily, when the HeLa cell nuclear extracts were precleared on GST-Spi-1 column before splicing reactions, their splicing activity was lost (Fig.  5). This suggests that the alteration of ␤-globin splicing by Spi-1/PU.1 occurred through a direct trapping of proteins involved in the splicing process. Altogether, these data suggest that Spi-1/PU.1 could interfere with splicing events.
The molecular mechanism by which the transcription factor Spi-1/PU.1 blocks the differentiation of proerythroblast and promotes their malignant transformation in the Friend erythroleukemia is not understood. Until now Spi-1/PU.1 was considered as a transcriptional regulator, targeting purine-rich DNA sequences in promoters and enhancers of some hematopoietic genes. The finding that Spi-1/PU.1 interacts with RNAbinding proteins and binds RNAs raises the possibility that in vivo, an elevated level of Spi-1/PU.1 may lead Spi-1/PU.1 to change the activities of RNA-binding proteins, like p54 nrb . Interestingly, some human sarcoma (21,22) and myeloid leukemia (23) are associated with chromosomal translocations that lead to the fusion of the two highly similar RNA-binding proteins TLS (14,24) and EWS (21), deleted in their RRM, with proteins that either mediate DNA binding like Fli-1 and Erg. Only the fusion proteins that exhibit altered RNA binding and transcriptional activities as compared to the native proteins (15,25) are oncogenic. It can be speculated that Spi-1/PU.1 normally binds DNA and regulates transcription of some hematopoietic genes through its cooperation with the transcrip-tional machinery. Unphysiological high concentration of Spi-1/ PU.1, consecutive to insertional mutagenesis, may promote its interaction with proteins involved in premRNA splicing. Although the function of p54 is unknown, its high homology with PSF and its ability to bind polypyrimidine sequences are indicative of a putative role in post-transcriptional modifications of RNA. Thus, Spi-1/PU.1 might disturb the post-transcriptional gene regulation by sequestering some RNA-binding proteins like p54 nrb . This alteration in alternative splicing, by preventing normal or promoting abnormal splicing complex formation, could be a mechanism involved in leukemogenesis.  1, 8, and 9) under splicing conditions in the absence or presence of 1 g of GST or GST fusion proteins. In lanes 8 and 9, HeLa cell nuclear extracts were first precleared on GST (lane 8) or GST-Spi-1 (lane 9) coated beads before the splicing reaction. Unspliced and spliced RNAs, separated on a 6% denaturing polyacrylamide gel, are indicated.