1 Department of Genetics, Case Western Reserve University, Cleveland, OH
44106-4955, USA
2 Department of Cell Biology and Molecular Genetics, University of Maryland,
College Park, MD 20742, USA
* Present address: Invitrogen Corporation, Carlsbad, CA 92008, USA
Author for correspondence (e-mail:
hks{at}po.cwru.edu)
Accepted 29 October 2002
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SUMMARY |
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Key words: Splicing regulation, SXL, SNF, U1 snRNP, U2AF, Drosophila
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INTRODUCTION |
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Some of the best understood examples of regulated splicing occur in
Drosophila melanogaster, where several tissue-specific
trans-acting factors have been identified
(Lopez, 1998). For example,
the female-specific RNA binding protein SEX-LETHAL (SXL) controls the splicing
pattern of the transformer (tra) pre-mRNA by binding to a
sequence adjacent to the regulated 3' splice site, thereby diverting
splicing to the female-specific splice site
(Granadino et al., 1997
;
Inoue et al., 1990
;
Sosnowski et al., 1989
;
Valcarcel et al., 1993
). SXL
also suppresses expression of the male-specific-lethal-2
(msl-2) gene by binding similar U-rich sequences
(Bashaw and Baker, 1995
;
Kelley et al., 1995
;
Zhou et al., 1995
). However,
the mechanism by which SXL regulates msl-2 processing is more complex
than for tra because it includes both translational repression and
splicing inhibition (Bashaw and Baker,
1997
; Forch et al.,
2001
; Gebauer et al.,
1999
; Gebauer et al.,
1998
; Kelley et al.,
1997
; Merendino et al.,
1999
). These two examples demonstrate that SXL is capable of
controlling expression of its target pre-mRNAs by diverse mechanisms.
In addition to controlling expression of tra and msl-2,
SXL also positively regulates its own expression to insure the continuous
production of SXL protein exclusively in females
(Bell et al., 1991;
Keyes et al., 1992
;
Sakamoto et al., 1992
). Tight
control of Sxl expression is crucial because the presence or absence
of SXL protein determines three major cell fate decisions: somatic sexual
differentiation, germline development and X-chromosome dosage compensation
(Cline and Meyer, 1996
;
Schutt and Nothiger, 2000
).
Thus, misregulation can result in sex-specific lethality, sex transformations
or ovarian tumors. Sxl expression is controlled in two phases. The
early phase is defined as the short period of time in early embryogenesis when
Sxl expression is first turned on in females; at this stage,
expression is controlled at the level of transcription
(Keyes et al., 1992
). The late
phase begins less than 1 hour later, when transcripts are detected in both
males and females (Keyes et al.,
1992
; Salz et al.,
1987
; Salz et al.,
1989
). Expression, however, remains sex specific because the
pre-mRNAs are differentially spliced such that protein-encoding mRNAs are
produced only in females (Bell et al.,
1988
; Samuels et al.,
1991
). The mRNAs produced in males all include the third exon,
which contains several inframe stop codons.
Sxl regulates its own expression through a mechanism by which the
female-specific SXL protein prevents inclusion of the translation-terminating
male specific exon (Bell et al.,
1991; Horabin and Schedl,
1993
; Sakamoto et al.,
1992
). Autoregulation has been linked to several essential
SXL-binding sites that are located in the introns on both sides of the
regulated male exon. Because these sites are located at some distance from the
5' and 3' intron/exon boundaries, it has been suggested that SXL
promotes exon skipping by interacting with and inactivating components of the
general splicing machinery (Horabin and
Schedl, 1993
). Indeed, recent studies carried out in tissue
culture cells suggest that SXL interacts with the general splicing factor
SPF45 at the male-exon 3' splice site to block inclusion at the second
catalytic step of splicing (Lallena et
al., 2002
). However, although blocking the 3' splice site is
by itself sufficient to ensure that the male exon is skipped in transient
tissue culture assays (Lallena et al.,
2002
; Penalva et al.,
2001
; Sakamoto et al.,
1992
), studies using similar splicing constructs expressed in
transgenic animals have shown that blocking the male exon 5' splice site
is also required for male-exon skipping
(Horabin and Schedl, 1993
).
Thus, the model proposed by Lallena et al.
(Lallena et al., 2002
) does
not provide a complete explanation for how SXL operates in the fly.
To date, the only general splicing factor for which there is in vivo
evidence for a regulatory role in the pathway leading to male-exon skipping is
sans-fille (snf), a protein component of the spliceosomal U1
and U2 snRNP particles (Flickinger and
Salz, 1994; Polycarpou-Schwarz
et al., 1996
; Stitzinger et
al., 1999a
). SNF was identified as a regulator of Sxl
splicing because, in females, the viable snf1621 mutation
disrupted the establishment of the Sxl autoregulatory splicing loop
in the germline, resulting in the accumulation of Sxl mRNAs spliced
in the male mode, leading to female sterility
(Bopp et al., 1993
;
Oliver et al., 1993
). Since
that time, the analysis of multiple snf alleles, including the lethal
null allele, has reinforced the view that snf functions as a
co-repressor of Sxl splicing in both the germline and the soma
(Albrecht and Salz, 1993
;
Cline et al., 1999
;
Flickinger and Salz, 1994
;
Hager and Cline, 1997
;
Salz, 1992
;
Salz and Flickinger, 1996
).
Furthermore, a central role for SNF in Sxl splicing regulation is
supported by its co-fractionation with SXL
(Deshpande et al., 1996
;
Samuels et al., 1998
). That
SNF is an integral snRNP protein has led to a model in which SXL blocks
male-exon use by interfering with snRNP function
(Deshpande et al., 1996
;
Salz and Flickinger, 1996
).
However, obtaining evidence in favor of this model has proven to be difficult
(Cline et al., 1999
), raising
the possibility that SNF acts outside of the snRNP in a manner analogous to
the way its human counterpart, U1A, inhibits its own polyadenylation
(Boelens et al., 1993
;
Klein Gunnewiek et al.,
2000
).
In this study, we characterize a viable snf mutation that interferes with both SXL complex formation, and assembly into the U1 snRNP. This analysis clarifies the role of snf in Sxl autoregulation, and suggests that SXL interacts with SNF in the context of the U1 snRNP. Consistent with this, we provide compelling in vivo evidence to link other U1 snRNP components to Sxl autoregulation by first showing that in embryonic extracts SXL can form an RNase-resistant complex with these factors, and then providing genetic evidence that supports the biological relevance of these physical interactions. Interestingly, we find that the interaction between the U1-70K protein and SXL does not require SNF, suggesting that SXL can interact with the U1 snRNP through several means. Using similar genetic and biochemical approaches, we also link SXL to the heterodimeric splicing factor U2AF. Together, these studies point specifically to a mechanism by which SXL antagonizes splicing during the early steps of spliceosome assembly by associating with these key splicing factors at both ends of the male exon.
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MATERIALS AND METHODS |
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Immunoprecipitation and GST pull down experiments
Immunoprecipitation, western blot analysis, RNA isolation from the
RNA-protein complexes and northern blot analysis were carried out as
previously described (Stitzinger et al.,
1999a).
For the GST pull-down assays, GST-tagged SXL protein was purified from
E. coli, using standard methods. The concentration of the GST fusion
protein was determined, and 60-80 µg of recombinant protein was combined
with 20 µl glutathione Sepharose 4B beads (Amersham Pharmacia Biotech AB)
and incubated for 1 hour 30 minutes at 4°C. After washing three times with
400 µl PBS to remove the unbound protein, the GST::SXL loaded beads were
combined with 100-150 µl embryonic extracts prepared from 400 µl 3- to
8-hour-old embryos homogenized in 1ml PBS containing protease inhibitor
cocktail (Roche). For experiments in which the extracts were pretreated with
RNase, 100 µl RNase A (10 mg/ml) and 50 µl RNase T1 (100,000 units/ml)
were added to 1 ml of extract and incubated for 30 minutes at 30°C. The
extract/bead mixture was incubated overnight at 4°C and then washed three
times with 400 µl PBS. To analyze the proteins selected in the pull-down
assays, 25 µl SDS loading buffer was added to the beads amd 20 µl loaded
onto a 12.5% SDS-polyacrylamide gel and analyzed by western blot analysis
using the following antibodies: anti-SNF
(Flickinger and Salz, 1994;
Habets et al., 1989
),
anti-U2AF38 (Rudner et al.,
1996
) and anti-U2AF50 (Rudner
et al., 1998
). A rabbit polyclonal antibody was raised against
amino acids 1-213 of the Drosophila U1-70K protein by standard
methods (Covance). Antibody binding was visualized using ECL (Amersham Life
Sciences). To analyze the RNAs selected in the pulldown assays, the RNAs were
isolated and analyzed by Northern blot analysis as described previously
(Stitzinger et al.,
1999a
).
To generate a homogeneous population of mutant embryos for the GST pull-down assays, embryos were collected as follows. For snf1621 and snf148, embryos were collected from snf/snf; P{w+; otu::Sxl} females crossed to snf males. For snfe8H, embryos were collected from snfe8H/snfe8H females crossed to snfe8H males (snfe8H is a fertile allele of snf). For snf5MER, which is a transgenic allele of snf, embryos were collected from snfJ210/snfJ210; P{w+; snf5MER}/P{w+; snf5MER}; P{w+; otu::Sxl} females crossed to snfJ210; P{w+; snf5MER}/P{w+; snf5MER} males.
RT-PCR analysis
The reporter construct and the sequences of the PCR primers used to amplify
the RNA expressed from the reporter constructs have been described previously
(Horabin and Schedl, 1993).
For the RT-PCR analysis, two procedures were used. In
Fig. 3, RNA from adults or
isolated ovaries was purified by standard methods. Reverse transcription was
carried out using the `Superscript First-Strand Synthesis System for RT-PCR'
(Gibco BRL) using 1-3 µg of RNA primed with random hexamers. The PCR
reactions were performed in a 100 µl volume with 2 µl of the RT
reaction, the Z1 lacZ primer and the Sxl-specific primer
using the `Expand High Fidelity PCR system' (Roche). The PCR conditions were
as follows: 95°C for 3 minutes; followed by 10 cycles of 95°C for 45
seconds, 62°C for 2 minutes, 68°C for 45 seconds. This was followed by
15 cycles of 95°C for 45 seconds, 62°C for 2 minutes, 68°C for 2
minutes 30 seconds, and a single final step at 68°C for 7 minutes. A 0.01%
aliquot of the first amplification reaction was then reamplified in a 100
µl volume using the Sxl-specific primer and the Z2 lacZ
primer internal to the one used in the first amplification reaction and 10
µCi 32P dCTP (3000 Ci/mmol, NEN) to label the products. The PCR
conditions were as follows: 94°C for 3 minutes; followed by 16 cycles of
95°C for 45 seconds, 61°C for 2 minutes, 72°C for 1 minutes 30
seconds; and a single step at 72°C for 5 minutes. Each PCR reaction (15
µl) was loaded on a 5% polyacrylamide gel and amplified fragments were
quantified using a phosphorimager. In Fig.
4, RNA was isolated from either adults or from embryos and the
RT-PCR reaction was carried out, using the same primers and the conditions
described elsewhere (Stitzinger, 1999b).
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RESULTS |
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Complex formation was assayed by pull down experiments in which a GST::SXL
fusion protein was expressed in bacteria, purified, bound to glutathione
sepharose beads and incubated with extracts made from either wild-type or
mutant embryos (Fig. 2). The
presence or absence of SNF in complexes formed on the beads was determined by
western blot analysis. Using this assay, we found that the GST::SXL fusion
protein, but not GST alone, could pull down SNF from extracts made from
wild-type embryos. As in similar studies
(Deshpande et al., 1996), we
found that this interaction is sensitive to RNase digestion (see
Fig. 4), and thus unlikely to
be direct. To test whether GST::SXL could pull down the mutant SNF proteins,
we made embryonic extracts from a homogeneous population of embryos whose only
source of SNF protein (both maternal and zygotic) was the mutant protein. As
illustrated in Fig. 2, we found
that GST::SXL was capable of selecting the SNF1621,
SNFe8H and SNF5MER mutant proteins from mutant extracts.
By contrast, GST::SXL could not pull down the SNF148 protein. We
have therefore identified a mutation that disrupts the association between SXL
and SNF.
snf148 is an uncharacterized mutation that was isolated
in a genetic screen for X-linked female-sterile alleles
(Swan et al., 2001).
Sequencing of the snf148-coding region revealed a single
missense mutation that changes a conserved asparagine at position 12 to an
aspartic acid (Fig. 1B).
According to the information from the crystal structures of the human U1A
protein bound to its RNA target sequence in the U1 snRNA
(Oubridge et al., 1994
) and
human U2B'' bound to the U2 snRNA
(Price et al., 1998
), the
conserved N12 residue contacts the RNA directly, suggesting that the
substitution of an aspartic acid at this position might disrupt assembly of
SNF148 into U1 snRNPs and/or U2 snRNPs. As in previous studies, we
assayed incorporation by testing whether U1 and U2 snRNAs can be
immunoprecipitated from whole cell extracts with antibodies directed against
SNF. Extracts from wild-type and mutant flies were incubated with anti-SNF
antibodies and the immunoprecipitated complexes tested for the presence of U1
and U2 snRNAs by northern blot analysis. As illustrated in
Fig. 1C, both U1 and U2 snRNAs
were immunoprecipitated from extracts made from wild-type,
snf1621 and snfe8H animals. In
extracts made from snf148 mutant animals, however, the SNF
specific antibody precipitated U2 snRNA without precipitating significant
amounts of U1 snRNA.
Thus, we have identified a single point mutation in the N-terminal RRM of
SNF that compromises both SXL complex formation and U1 snRNP incorporation,
without having an apparent effect on U2 snRNP incorporation. The fact that
snf148 animals are viable indicates that the stable
association of SNF with the U1 snRNP complex is not crucial for U1 snRNP
function in vivo. Indeed, recent biochemical studies have also suggested that
SNF is dispensable for U1 snRNP function
(Labourier and Rio, 2001).
Sxl splicing autoregulation is disrupted in
snf148 mutant females
We had anticipated that a lack of association between SNF and SXL would
cause a major perturbation of splicing autoregulation, resulting in the
accumulation of Sxl mRNAs spliced in the male mode, an outcome known
to result in female lethality. Instead, we found that
snf148 mutant females were simply sterile.
To examine the snf148 mutant phenotype in more detail, ovaries from both wild-type and mutant females were fixed and stained with SNF antibodies as well as DAPI to visualize the nuclei. In wild-type ovaries, each ovariole consists of a series of egg chambers, each of which contains 15 polyploid nurse cells and an oocyte (Fig. 3A). As in the soma, SNF is localized to the nucleus in the early stages of oogenesis, including the region at the tip of the ovariole, called the germarium (Fig. 3C). Egg chambers from snf148 mutant females, however, are filled with many small nuclei (Fig. 3B). This defect, which appears to be identical to the ovarian phenotype of other female-sterile alleles of snf, is called an ovarian tumor phenotype. This experiment also demonstrates that the SNF148 mutant protein retains its ability to localize to the nucleus (Fig. 3D), eliminating the trivial explanation that the female-sterile mutant phenotype is a result of SNF protein mislocalization.
Because a common characteristic of the snf ovarian tumor mutant
phenotype is the presence of male-specific Sxl RNA products, we
examined the Sxl splicing pattern in isolated ovarian tissue from
snf148 mutant animals. In these studies, we used the
Sxl reporter construct described elsewhere
(Horabin and Schedl, 1993),
which contains Sxl genomic sequences from exon L2 to the middle of
exon L4 fused to lacZ-coding sequences and faithfully reproduces the
endogenous splicing pattern (Fig.
3E). When Sxl spliced products were analyzed by
semi-quantitative RT-PCR in wild-type adults, expression of this reporter
construct mimics the sex-specific regulated splicing of the endogenous locus:
In males, the reporter was spliced to include exon L3, generating an L2-L3-L4
product, while in females the reporter was spliced to exclude exon L3,
generating an L2-L4 spliced product that is 200 bp shorter
(Fig. 3F, lane 1 and 2).
Similarly, we found that the male-specific exon is consistently skipped in
isolated ovarian tissue (lane 4). By contrast, two products were detected in
ovaries isolated from snf148 mutant animals. One
corresponds to the L2-L3-L4 male-specific product and the other corresponds to
the L2-L4 female-specific product (lane 5). Thus, we conclude that the
Sxl male-exon is not reliably skipped in snf148
mutant ovaries. Importantly, we could rescue the mutant females to fertility
by expression of a transgenic copy of the Sxl cDNA under control of a
germline-specific promoter P{otu::SxlcDNA}, demonstrating that the
perturbation of Sxl splicing is responsible for the sterile phenotype
(data not shown).
The viability of snf148 mutant females could be
explained if the maternally produced SNF protein provided by their
heterozygous mothers was sufficient for the successful establishment of the
Sxl autoregulatory loop in the mutant embryos. To test this
possibility, we assessed the viability of snf148 mutant
animals whose only source of SNF protein, both maternal and zygotic, was
derived from the mutant allele. To generate these animals, we bypassed the
sterility of the snf mutant females by expression of the
germline-specific transgene P{otu::SxlcDNA}. Previous studies have
shown that this transgene drives Sxl expression exclusively in the
germline, and does not interfere with our assessment of the effects of
snf on zygotic Sxl expression
(Salz and Flickinger, 1996).
Contrary to our expectations, the survival rate of snf148
mutant females was equivalent to snf148 mutant males
(n>500), and comparable with the survival rate of animals from
heterozygous mothers. Moreover, in accordance with their viable phenotype,
only a small amount of male-specific Sxl product was detectable in
RNA isolated from these females (Fig.
3F, lane 3). Thus, we conclude that the snf148
mutation does not have a major effect on either Sxl splicing in
embryos or female viability in an otherwise wild-type background.
Although our data indicate that snf is dispensable for the establishment of Sxl autoregulation in the embryo, they do not imply that snf has no role in Sxl autoregulation. In fact, its involvement is evident when we test for female-viability in a genetically sensitized background. For example, when snf mutant females (snf148/snf148; P{otu::SxlcDNA}) were crossed to males carrying the normally recessive null allele of Sxl (Sxl7B0), only 6% of her Sxl7BO/+ daughters survive.
SXL associates with U1 snRNP particles in whole cell extracts
The finding that mutation of a single residue in the N-terminal RRM of SNF
interferes with both SXL complex formation and assembly into the U1 snRNP
suggests that SXL associates with SNF in the context of an intact U1 snRNP. To
address this possibility further, we tested whether SXL can physically
associate with the U1 snRNP in embryonic extracts. Using GST pull-down assays,
we found that both the U1 snRNA (data not shown) and U1-70K protein
(Fig. 4A) could be selected
from embryonic extracts by GST::SXL but not by GST alone, thus demonstrating
that SXL can associate with the intact U1 snRNP particle. Interestingly, of
the two U1-70K isoforms observed in whole cell extracts, only the more rapidly
migrating U1-70K species was identified in the pull-down experiments. As
U1-70K is known to be phosphorylated (Tazi
et al., 1993; Woppmann et al.,
1990
), we suspected that the more rapidly migrating form might be
dephosphorylated. Consistent with this notion, we found that phosphatase
treatment of the embryonic extracts prior to western blot analysis resulted in
a single U1-70K species with a similar mobility to the protein detected in the
pull down experiments (data not shown). Thus, U1-70K phosphorylation may
modulate SXL complex formation.
To determine whether the interaction between SXL and U1-70K is mediated by RNA present in the extract, we pretreated the extract with RNase prior to performing the GST pull-down assays. The results show that, in contrast to the SXL/SNF interaction, the interaction between SXL and U1-70K is resistant to RNase digestion (Fig. 4A). Thus, the SXL/U1-70K interaction is unlikely to be mediated by an RNA, although we cannot exclude the possibility that a bridging RNA (e.g. U1 snRNA) was protected from the nuclease.
The difference in RNase sensitivity between the SXL/SNF and SXL/U1-70K complex under these conditions suggests that the association between U1-70K and SXL is not mediated by SNF. To test this directly, pull-down assays were used to determine if GST::SXL could select U1-70K from snf148 mutant extracts. As illustrated in Fig. 5B, U1-70K was selected from these mutant extracts, demonstrating that the association between U1-70K and SXL does not depend on SNF.
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Mutations in U1-70K enhance Sxl mutations, resulting in
female-lethality and Sxl splicing defects
The data presented thus far indicate that SXL can physically associate with
the U1 snRNP. If SXL works together with the U1 snRNP to promote male-exon
skipping, each component of the U1 snRNP might, therefore, have an independent
effect on the function of the SXL-blocking complex. This predicts that
simultaneously lowering the available SNF and U1-70K protein in the embryo
might have an additive effect, which will reduce the efficiency of male-exon
skipping, resulting in female-lethality.
To test this idea, we asked whether we could detect a maternal-effect genetic interaction phenotype between null alleles of snf and U1-70K (Fig. 5A). In control crosses, we find that snfJ210/+ mothers provide a sensitized genetic background for these assays because only 81% of her Sxl7B0/+ daughters survive. Reducing the dose of U1-70K, however, has no effect, as the viability of Sxl7B0/+ daughters from U1-70K1/+ mothers was not significantly reduced (data not shown). However, when the mothers were heterozygous for both snfJ210 and U1-70K1, there was a significant reduction in viability, with only 34% of the expected Sxl7B0/+ daughters surviving.
To determine whether this synergistic female lethality can be correlated with an increase in male-exon inclusion, we assayed splicing using an X-linked Sxl reporter construct linked to the same chromosome as the Sxl7B0 mutant allele (Fig. 5B). As a consequence of this genetic linkage, the Sxl7B0/+ female progeny will carry the reporter construct and their male siblings will not. In control experiments, when the spliced products were analyzed by RT-PCR, only the female-spliced product was detectable in embryos from snfJ210/+ mothers (lane 3). By contrast, we observed a significant amount of the male-spliced product in embryos from snfJ210/+; U1-70K1/+ mothers (lane 5), consistent with the synergistic female-lethal phenotype. These additive gene-dose effects, therefore, are consistent with a model in which SXL works together with the U1 snRNP to block male-exon inclusion.
Genetic and physical interactions between U2AF and SXL
While the preceding data implicate components bound to the male exon
5' splice site in Sxl splicing autoregulation, recent studies
have suggested that additional components bound to the male-exon 3'
splice site are also important (Penalva et
al., 2001). Moreover, in studies carried out in HeLa cells, the
heterodimeric splicing factor U2AF was crosslinked to the intron on the
upstream side of the Sxl male exon
(Lallena et al., 2002
). To
obtain functional evidence to support a role for U2AF in Sxl
autoregulation, we used the genetic and biochemical assays described in the
previous section.
First, we showed that loss-of-function mutations in the small subunit of
U2AF (U2af38E18) and snf exert synergistic
effects on the viability of Sxl7B0/+ females and on
Sxl splicing (Fig.
5A). Only 21% of the expected Sxl7B0/+
heterozygous females were recovered from
snfJ210/+; U2af38
E18/+
mothers. Furthermore, we found that the female lethality was correlated with
Sxl splicing defects, as a significant amount of male-spliced product
is detected in Sxl/+ female embryos collected from
snfJ210/+; U2af38
E18/+
mothers (Fig. 5B, lane 4).
Together, these in vivo findings indicate that the small subunit of U2AF plays
an active role in Sxl splicing autoregulation.
Interestingly, we did not detect a genetic interaction with null mutations in the large subunit of U2AF (U2af50X15). However, as dose-sensitive interactions are detectable only if the gene product being assayed is present in limiting quantities, the failure to detect a genetic interaction may simply mean that reducing the level of this core splicing factor is not sufficient to compromise Sxl splicing significantly. Thus, while detection of a genetic interaction provides compelling evidence for the active involvement of the small subunit of U2AF in Sxl splicing regulation, the failure to detect an interaction does not argue against a role for the large subunit of U2AF.
Finally, to support the genetic role of U2AF in Sxl splicing autoregulation, we tested whether the U2AF heterodimer could be selected from embryonic extracts by GST::SXL in pull-down assays. As illustrated in Fig. 6, our data indicate that both subunits of U2AF associate with SXL. This association is robust, as we found that GST:: SXL could pull down both the large and small subunit from extracts pretreated with RNase (data not shown). Moreover, we could pull down both subunits of U2AF from snf148 mutant extracts, demonstrating that the association between these two proteins does not require a prior association between SXL and SNF. Together with the genetic interaction data, these physical associations suggest that SXL blocks use of the male-exon 3' splice site by associating with the U2AF heterodimer.
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DISCUSSION |
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The Sxl male exon is unusual in that it contains two 3' AG
dinucleotides separated by a short polypyrimidine tract
(Bell et al., 1988).
Interestingly, although the upstream 3' splice site is used almost
exclusively for exon ligation in tissue-culture cells, both 3' splice
sites are required for SXL-mediated male-exon skipping
(Penalva et al., 2001
).
Moreover, crosslinking studies in HeLa cell extracts have shown that the U2AF
heterodimer binds to the downstream 3' splice site and the intervening
polypyrimidine tract (Lallena et al.,
2002
), suggesting that U2AF may play an active role in
Sxl regulation. We validate these biochemical data by demonstrating
that the SXL protein can associate with the Drosophila U2AF
orthologs. More importantly, our genetic data provide compelling support for
the biological relevance of these interactions by demonstrating that in
females, the small subunit is important for both Sxl male-exon
skipping and female viability. In addition to demonstrating a role for U2AF in
Sxl autoregulation, this genetic result is notable because previous
studies have failed to find RNA splicing defects associated with small subunit
mutations (Burnette et al.,
1999
; Rudner et al.,
1996
). Whether our success reflects substrate-specificity or
sensitivity of our assay remains to be determined.
In addition to controlling the use of the male exon 3' splice site, our studies suggest that SXL controls the use of the male-exon 5' splice site by interacting with the U1 snRNP. We were able to establish this connection in three ways. First, we find that mutation of a single residue in the N-terminal RRM of SNF compromises both complex formation with SXL and assembly into the U1 snRNP, thus suggesting that the two events are linked. Second, we demonstrated that, in addition to SNF, SXL can associate with other integral U1 snRNP components, including the U1-70K protein and the U1 snRNA in whole cell extracts. Finally, our genetic interaction data provide evidence that U1-70K, like SNF, is important for the successful establishment of the Sxl autoregulatory splicing loop in females.
Although our discovery that SNF is an snRNP protein was the first clue that
SXL might act by associating with components of the general splicing
machinery, the role of SNF has remained enigmatic. We clarify the role of SNF
by demonstrating that its contribution to the function of the U1 snRNP is not
absolutely essential for viability of either sex, and that SXL can associate
with the U1 snRNP through a SNF-independent mechanism. Nevertheless, our in
vivo analysis continues to support a role for snf in Sxl
splicing autoregulation by demonstrating that Sxl splicing defects
are detectable under specific conditions. Interestingly, the phenotypic
consequences of these Sxl splicing defects are more severe in the
germline than in the soma. One possible explanation for this difference is
that the requirements for Sxl splicing autoregulation are
fundamentally different in the two tissue types. We think, however, that it is
more likely that the mechanism is the same, but that the additional
interaction with the U1 snRNP provided by SNF becomes critical when SXL
protein levels are low. This hypothesis is based on the fact that, in the
germline, the majority of SXL protein is cytoplasmic, and thus low levels of
nuclear SXL protein are the norm (Bopp et
al., 1993). By contrast, in other tissues, the SXL protein
accumulates in the nucleus, enabling the SXL-U1 snRNP complex to form even
when SNF is not stably associated with the U1 snRNP. Our finding that these
snf mutant females rarely survive if they are also heterozygous for
Sxl, provides additional support for the idea that SNF function is
only critical when SXL protein levels are low.
Together, our studies argue that interactions between SXL, the U1 snRNP and U2AF underlie the mechanism by which SXL promotes skipping of the male exon. Based on these studies, we propose a model in which SXL acts not by preventing assembly of the U1 snRNP or U2AF onto the pre-mRNA, but instead interacts with the U1 snRNP bound to the male-exon 5' splice site, and U2AF at the male-exon 3' splice site, to form complexes that block these general splicing factors from assembling into a functional spliceosome (Fig. 7). These 5' and 3' SXL blocking complexes might function independently or they might interact across the exon to form a larger inhibitory complex. Furthermore, because we have been unable to demonstrate that SXL interacts directly with either U1-70K or U2AF, we speculate that one or more bridging proteins are required to link SXL to the general splicing machinery.
|
Although our in vivo approach cannot directly address when in the pathway
of spliceosome assembly SXL acts, biochemical studies have shown that during
the course of spliceosome assembly, U2AF and the U1 snRNP are only transiently
associated with splicing substrates, and are released before the formation of
a functional spliceosome. Therefore, based on our studies, it seems reasonable
to propose that SXL acts by blocking splicing after splice site recognition
but before catalysis begins. Our data are therefore difficult to reconcile
with the recent model presented by Lallena et al.
(Lallena et al., 2002), which
proposes that SXL blocks splicing after spliceosome assembly, at the second
catalytic step of the reaction. Using RNA interference in Drosophila
tissue culture cells, Lallena et al.
(Lallena et al., 2002
)
demonstrate that efficient male exon skipping depends on the presence of
SPF45, a protein that is known to be required for the second step of splicing.
Together with studies that show that SPF45 can bind to the upstream 3'
splice site of the Sxl male exon and physically interact with SXL,
these data point to a role for SPF45 in Sxl splicing regulation.
However, the primary evidence that SXL blocks the splicing reaction during the
second step rests on the results of in vitro splicing assays in which SXL was
shown to inhibit splicing of a chimeric splicing substrate that contains only
a small region of the intronic region required for successful autoregulation
in vivo (Horabin and Schedl,
1993
). We suspect that by looking at this 48 bp region, which
contains a dispensable SXL-binding site in addition to the two potential
3' splice sites, out of context, Lallena et al.
(Lallena et al., 2002
) have
uncovered a failsafe mechanism that comes into play when SXL-mediated splicing
regulation is otherwise compromised. Additional studies investigating the
function of SPF45 in vivo will be required to determine the importance of this
second step blocking mechanism and should provide insight into whether
multiple mechanisms are needed to drive efficient regulated exon skipping.
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ACKNOWLEDGMENTS |
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