(Received for publication, February 22, 1996; and in revised form, March 22, 1996)
From the
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
p54 (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 p54
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 5`-GGAA-3` 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(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.
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. ()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
(54 kDa)(9) .
p54
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).
Figure 1:
In
vitro association of p54 with Spi-1/PU.1. A, schematic diagram of Spi-1, Fli-1 and p54
functional domains fused to GST. AD, activation domain; PEST, domain rich in proline, glutamic acid, serine, and
proline; DBD, DNA-binding domain; RRM,
RNA-recognition motif. B, interaction of p54
with Spi-1/PU.1 and Spi-B proteins.
S-Labeled in
vitro translated Spi-1, Spi-B, Fli-1, and Ets-2 proteins were
mixed with GST-p54
or GST absorbed on
glutathione-Sepharose beads. Complexes were washed, eluted with
SDS-sample buffer, and analyzed by SDS-PAGE. 10% input proteins are
shown. Molecular mass markers are indicated on the right. C, mapping of the domains involved in interactions between
Spi-1/PU.1 and p54
. GST pull-down assays of
S-labeled in vitro translated Spi-1/PU.1 and
p54
proteins were performed with the functional domains
of Spi-1/PU.1, Fli-1, and p54
fused to GST. Input
proteins are shown. Molecular mass markers are indicated on the right.
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 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
. GST-p54
fusion
protein was incubated in the presence of
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
, revealing that in vitro association of
p54
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 was mapped by testing interactions of
S-labeled in vitro translated p54
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
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
. 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
, like
PSF(17) , contains a central RNA binding domain with two RNA
recognition motifs (RRM). Different truncated GST-p54
fusion proteins (Fig. 1A) were tested for their
abilities to bind in vitro
S-translated
Spi-1/PU.1 protein. Interactions occurred only between the entire
GST-p54
protein or the GST-RBD-p54
,
suggesting that p54
bound Spi-1/PU.1 by its RNA binding
domain. The RNA binding activities of GST-p54
and
GST-RBD-p54
were controlled on a Northwestern blot probed
with
P-labeled poly(A)
mRNAs from 745A
cells (Fig. 4A). Altogether, these data suggested that
the interaction of the transcription factor Spi-1/PU.1 with the
RNA-binding protein p54
involved their respective DNA-
and RNA-binding domains.
Figure 4:
Spi-1/PU.1 binds RNA. A, GST
fusion proteins were separated by gel electrophoresis and blotted onto
nitrocellulose. The blot was stained with Ponceau Red (left
panel) and then probed with P-end-labeled
poly(A)
RNAs (right panel). Molecular mass
markers are indicated on the right. B, RNA binding
activities of Spi-1/PU.1, Fli-1, and Ets-2.
S-labeled in vitro translated proteins were bound to homoribopolymers
linked to agarose beads. 50% input are shown. Molecular mass markers
are indicated on the right. C, EMSA of in vitro translated Spi-1/PU.1 incubated with
P-labeled
c-Fes-RE probe in the presence of homoribopolymer
competitor.
Then, we searched whether p54 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
(tag-Myc-p54
). 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
expression
vectors. This provided evidence that Spi-1/PU.1 was associated with
p54
in vivo.
Figure 2:
Coimmunoprecipitation of Spi-1/PU.1 and
p54. Nuclear extracts from COS cells expressing
tag-Myc-p54
and/or Spi-1/PU.1 proteins were
immunoprecipitated by anti-Myc epitope antibodies. The
immunoprecipitated proteins were analyzed by Western blotting with
either anti-Myc or anti-Spi-1/PU.1 antibodies (7) using the ECL
procedure (Amersham). The asterisk indicates the position of
immunoglobulin G heavy chains.
The interaction between the DBD
of Spi-1/PU.1 and the RBD of p54 suggested that it could
alter the function of each partner. p54
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
. The pyrimidine-rich sequence
of the branchpoint/polypyrimidine tract RNA, which is part of most 3`
splice sites in mammalian introns, is targeted by PSF(17) .
Since p54
presents 70% identity with PSF in its RRM, we
tested whether p54
could bind such RNA sequence. We
observed (Fig. 3) that, like PSF, p54
binds the
pyrimidine-rich sequence of the 3` 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
reduced the
formation of the GST-p54
-RNA complex in a dose-dependent
manner, revealing that Spi-1/PU.1 impedes the binding of p54
to RNA.
Figure 3:
Spi-1/PU.1 inhibits the RNA binding
activity of p54. Binding of p54
to the RNA
pyrimidine-rich sequence in the 3` splice site of the first
-tropomyosin intron. Amounts of GST or GST-Spi-1 added in EMSA are
indicated. The right panel shows competition experiments
performed with 250 ng of poly(A), poly(C), poly(G), and
poly(U).
The binding of Spi-1/PU.1 to the RNA-binding protein
p54 prompted us to check whether Spi-1/PU.1 binds RNA.
Various deletion mutants of Spi-1/PU.1 and p54
fused to
GST were analyzed by Northwestern blot using the labeled
poly(A)
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
. 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
E3`(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
exhibited
affinity for poly(G) and poly(U) revealing that Spi-1/PU.1 and
p54
display a similar ability to bind poly(G). In
agreement with the absence of in vitro-interaction between
p54
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 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.
Figure 5:
Spi-1/PU.1 alters the splicing of a
-globin minigene in vitro.
P-Labeled
-globin mRNA was incubated with HeLa cell nuclear extracts (except lanes 1, 8, and 9) under splicing conditions
in the absence or presence of 1 µg 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.
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 RNA-binding 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. 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 transcriptional
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
. This alteration in
alternative splicing, by preventing normal or promoting abnormal
splicing complex formation, could be a mechanism involved in
leukemogenesis.