Cell, Vol. 74, 105-114,
July 16, 1993, Copyright
0 1993 by Cell Press
A Splicing Enhancer Complex
Controls Alternative Splicing
of doublesex Pre-mRNA
Ming Tian and Tom Maniatis
Harvard University
Department of Biochemistry and Molecular
Cambridge, Massachusetts 02138
Biology
Female-specific
splicing of Drosophila doublesex (dsx)
pre-mRNA is regulated by the products of the transformer (fra) and transformer
2 (fra2) genes. In this paper we show that Tra and Tra2 act by recruiting general
splicing factors to a regulatory element located downstream of a female-specific
3’splice site. Remarkably,
Tra, TraP, and members of the serinelarginine-rich
(SR) family of general splicing factors are sufficient to
commit dsx pre-mRNA to female-specific
splicing, and
individual SR proteins differ significantly
in their ability
to participate in commitment complex formation. Characterization
of the proteins associated
with affinity
purified complex formed on dsx pre-mRNA reveals
the presence of Tra, Tra2, SR proteins, and additional
unidentified
components.
We conclude that Tra, Tra2,
and SR proteins are essential components
of a splicing
enhancer complex.
Alternative splicing of individual nuclear pre-messenger
RNAs (pre-mRNAs) can lead to the production of multiple
mRNAs encoding functionally distinct proteins (for recent
reviews see Maniatis, 1991; Green, 1991; Nadal-Ginard
et al., 1991; Rio, 1992). A striking example of this phenomenon is the sex determination pathway of Drosophila melanogaster, which involves a cascade of regulated splicing
events (for reviews see Baker, 1989; Steinmann-Zwicky
et al., 1990). Alternative splicing of doublesex (dsx) premRNA, the last step in this regulatory hierarchy, generates
male or female-specific
mRNAs. In females, dsx premRNA, which contains six exons, is spliced to produce a
dsx mRNA consisting of exons 1, 2, 3, and 4 (Figure 1;
Burtis and Baker, 1989) and this mRNA encodes a transcriptional repressor of male sexual differentiation (Baker
and Ridge, 1980; Nothiger et al., 1987). In contrast, male
dsx mRNA is composed of exons 1,2,3,5,
and 8 (Figure
1; Burtis and Baker, 1989), and it encodes a protein that
represses the expression of genes required for female
development
(Baker and Ridge, 1980; Nothiger et al.,
1987).
Regulation of dsx pre-mRNA splicing involves positive
control by Tra and Tra2 of the female-specific
3’ splice
site immediately upstream of exon 4 (Hedley and Maniatis,
1991; Hoshijima et al., 1991; Ryner and Baker, 1991; Tian
and Maniatis, 1992). The pyrimidine tract of the femalespecific 3’splice site, an important determinant of splicing
efficiency (Green, 1991) is interrupted by purines (Burtis
and Baker, 1989). The male-specific 3’ splice site at exon
5 conforms to the consensus. As a result, the male-specific
3’ splice site is used by default in males. In females, Tra
and Tra2 activate the female-specific 3’splice site, leading
to the female splicing pattern.
Both Tra (Boggs et al., 1987) and Tra2 (Amrein et al.,
1988; Goralski et al., 1989) contain motifs characteristic
of proteins involved in RNA processing. Tra consists of an
extended serinelarginine-rich
domain (SR domain), while
Tra2 contains two SR domains and a ribonucleoprotein
consensus-type RNA recognition motif (RRM) (Bandziulis
et al., 1989). Both the SR domain and RRM are also present in members of the SR family of splicing factors (Ge
et al., 1991; Krainer et al., 1991; Fu and Maniatis, 1992a;
Zahler et al., 1992) and in splicing factors U2AF (Zamore
et al., 1992) Ul 70K protein (Theissen et al., 1988;
Mancebo et al., 1990), and suppressor of white apricot
(su(vP)) protein (Chou et al., 1987). Tra, Tra2, and several
unidentified nuclear proteins bind specifically to a regulatory element (the repeat element) in the female-specific
exon(Hedleyand
Maniatis, 1991; Tianandfvlaniatis,
1992;
lnoue et al., 1992). This element is located about 300 nt
downstream of the female-specific 3’ splice site and contains six copies of a 13 nt repeat sequence (Nagoshi and
Baker, 1990). The repeat element is both necessary and
sufficient for mediating Tra- and Tra2-dependent
regulation of splicing (Nagoshi and Baker, 1990; Hedley and
Maniatis, 1991; Hoshijima et al., 1991; Ryner and Baker,
1991; Tian and Maniatis, 1992).
In this paper, we study the mechanisms of Tra and Tra2
function using a previously established in vitro system in
which the dsx female-specific
splicing can be activated
by recombinant Tra and Tra2 (Tian and Maniatis, 1992).
We show that Tra and Tra2 cooperate with nuclear factors
to commit dsx pre-mRNA to the splicing pathway, and we
identify the factors involved. Moreover, we use an affinity
purification method to demonstrate that Tra and Tra2 act
by recruiting general splicing factors to a regulatory sequence located downstream of the female-specific splice
site. We propose that Tra and Tra2 activate dsx femalespecific splicing by promoting the formation of a splicing
enhancer complex.
Results
Tra and Tra2 Commit dsx Pre-mRNA to
Female-Specific
Splicing
Since Tra and Tra2 function as a binary switch in splice
site choice, they most likely act at the early commitment
stage of spliceosome assembly. To investigate this possibility, we designed a functional assay for dsx commitment
complex (dsxCC) formation that involves competition and
preincubation
experiments.
In the competition experiments, we added unlabeled
RNAs with or without the repeat sequence to in vitro splicing reactions in which dsxfemale-specific
splicing can be
activated by recombinant Tra and Tra2 (Van and Maniatis,
1992; Figure 2, lanes 2 and 3). Since Tra and Tra2 bind
Cell
106
Figure
1. The Sex-Specific
Splicing
Pattern
of dsx Pre-mRNA
The open boxes, hatched box, and closed boxes represent
common
exons, the female-specific
exon, and male-specific
exons, respectively. The exon numbers are indicated, and the lines connecting
the
exons represent splicing. pA at the end of exon 4 and exon 6 represents
the site for cleavage and polyadenylation.
specifically to the repeat element, repeat-containing
competitor RNAs should titrate away these factors and abolish
Tra- and Tra2-dependent
regulation. Consistent with this
prediction, the addition of repeat-containing
RNA competitors inhibits the female-specific splicing (Dl , D4, and D5;
Figure 2, lanes 10-16) while RNAs without the repeat
sequence have no effect (D3 and D6; Figure 2, lanes
4-6 and 17-l 6). The competitor RNA containing just one
copy of the repeat sequence weakly inhibits the femalespecific splicing (D2; Figure 2, lanes 7-9).
Based on these results, we designed a commitment
complex assay in which labeled dsx Dl substrate was
preincubated
with Tra, Tra2, and micrococcal nuclease
(MN)-treated nuclear extract (Krainer and Maniatis, 1965).
MN-treated extract is splicing deficient because it lacks
functional small nuclear RNAs (snRNAs), but presumably
contains all the protein components necessary for splicing. The reason for including MN-treated extract in the
preincubation
stems from our previous observation that
Tra and Tra2 promote the specific binding of several nuclear proteins to the repeat element (Tian and Maniatis,
1992). The preincubation was followed by the addition of
a splicing-competent
nuclear extract plus an excess of
unlabeled repeat-containing
competitor RNA (D5), and the
incubation was continued. If during the preincubation the
binding of Tra, Tra2, and the nuclear proteins to repeat
element results in the formation of a stable complex, the
substrate will be spliced in the presence of the repeatcontaining competitor RNA during the second incubation
period.
After preincubation
with Tra, Tra2, and MN-treated extract, female-specific splicing was indeed observed in the
presence of repeat-containing
competitors (Figure 3, lane
5). Thus, a stable complex that is resistant to competition
was formed during preincubation.
Since this stable complex commits the dsx pre-mRNA to the female-specific
splicing pathway, we refer to it as the dsx commitment
complex (d.sxCC). Tra and Tra2 are required for dsxCC
formation, since no splicing was observed when they are
absent during the preincubation
step (Figure 3, lane 3).
However, Tra and Tra2 are not sufficient for dsxCC formation, since preincubation of the Dl substrate with Tra and
Tra2 in the absence of MN-treated nuclear extract does
not lead to &WCC formation (Figure 3, lane 4). dsxCC
formation therefore requires both Tra and TraP and addi-
d.sxm
I
------
4
I
02
03
D4
05
D6
Figure 2. Repeat-Containing
vation of dsx Female-Specific
RNAs Competitively
Inhibit In Vitro ActiSplicing by Tra and TraP
AYP-IabeleddsxRNAsplicingsubstratecontaining
therepeatelement
(Dl) was incubated in a HeLa cell nuclear extract in the absence (lane
2) or presence
(lane 3) of recombinant
Tra and TraP. The reaction
products
were then fractionated
on a denaturing
polyacrylamide
gel
and visualized by autoradiography.
To identify the dsx RNA sequences
required for splicing, excess unlabeled competitor
RNA containing
various regions of dsx pm-mRNA (Dl-D6)
was added to the reaction
at the same time as the labeled precursor.
The competitor
RNAs are
diagrammed
below the autoradiogram.
The splicing substrate
is Dl.
The precursor
and the splicing product are indicated at the right of
the autoradiogram.
Lane 1, unspliced Dl substrate;
lane 2, incubation
in the absence of Tra and TraP; lanes 3-18, splicing reactions carried
out in the presence of Tra and Tra2 and different amounts of competitor
RNAs as indicated above each lane. Minus indicates no competitor
added. In the three titration points for D3, D2, and Di (lanes 4-12)
the ratios of competitor
to substrate are 1O:l ,50:1, and 250:1, respectively. In the two titration points for D4, D5, and D6 (lanes 13-16) the
ratios of competitor
to substrate are 5O:l and 250:1, respectively.
tional proteins present in the MN-treated nuclear extract.
The presence of repeat-containing
RNA competitors
during preincubation
abolished dsxCC formation, while
the presence of nonspecific RNA had no effect (Figure 3,
lanes 6-7). Thus, &WCC formation requires the factors
binding to the repeat element. Significantly, the formation
of the &WCC does not require ATP (Figure 3, lane 6).
In mammalian systems, the formation of E complex, the
earliest known prespliceosome,
is also ATP independent
(Michaud and Reed, 1991). However, E complex contains
Ul small nuclear RNP (snRNP). The formation of the
dsxCC in MN-treated extract, which lacks fully functional
Ul snRNP, suggests that the formation of the dsxCC may
precede E complex assembly. However, we can not eliminate the possibility that E complex and the dsxCC may
be formed independently.
;;;itive
Regulation
of dsx Pre-mRNA
TraiTraP
D5
1 tlr
4
NEm
Tra+Tra*
s
2 h,
NE
Lx
f h,
5
s
2 llr
Tra+TraP
NE
D5
2 tlr
1 hr
6
NEm
s
ing sequential precipitation with ammonium sulfate and
MgCI? (Zahler et al., 1992). We carried out this purification
procedure and followed the activities that complement Tra
and Tra2 in dsxCC formation (Figure 4).
After ammonium
sulfate precipitation
of HeLa cell
whole-cell extracts, SR proteins are present in the 65%90% saturated fraction (Zahier et al., 1992). This fraction
also complemented Tra and Tra2 in the commitment complex assay (Figure 4, lane 6). We then precipitated the SR
proteins from the 65%~90% ammonium sulfate-saturated
fraction with MgCl2. Significantly, the complementing
activity is contained exclusively in the pellet fraction (Figure
4, lanes 7-6). When analyzed by SDSpolyacrylamide
gel
eiectrophoresis (SDS-PAGE),
the pellet fraction contains
the characteristic set of SR proteins and only low levels
of contaminating
non-SR proteins (Figure 4, lane SR;
NE
s
NP
Splicing
~ra+Tra*
D6
NE
D5
ikDI
M
94-
ui,
ST--
a&
SR
I*
- sl3p75
u*
-sip55
43 -
Figure
3. Tra and Tra2 Are Required
for the Formation
of the dsxCC
This figure shows the results of a splicing assay used to identify factors
required for the formation of a dsxCC on 32P-labeled Dl RNA. Analysis
of the precursor
and splicing products was as described
in Figure 2.
Lane 1, unspliced substrate;
lane 2, incubation
in nuclear extract in
the presence of Tra and TraP; lanes 3-8, preincubation
experiments,
with the order of addition of various factors diagrammed
below the
autoradiogram.
In these schemes,
the horizontal lines represent
the
progress
of the reactions,
and the left ends are the beginning
of
the reactions.
The addition order of reagents and incubation
times
are indicated above the lines. NE, nuclear extract; NE”, MN-treated
nuclear extract; S, splicing substrate;
CP, creatine phosphate.
Members of the SR Family of General Splicing
Factors Are Required for dsxCC Formation
We used dsxCC formation as a functional assay to identify
the nuclear proteins that complement Tra and TraP in the
preincubation assay. The chromatographic
behavior of the
complementing
activities in our initial purification efforts
suggested that they may correspond to a group of known
splicing factors, the SR proteins (Ge et al., 1991; Krainer
et al., 1991; Fu and Maniatis, 1992a; Zahler et al., 1992).
The SR proteins are characterized by the presence of an
SR domain in the C-terminal half of the protein and an FIRM
in the N-terminal half. These proteins can complement
splicing-incompetent
SlOO extracts, and they can infiuence splice site choice in pre-mRNAs containing competing 5’or 3’spiice sites (Ge et al., 1991; Krainer et al., 1991;
Mayeda et al., 1992; Fu et al., 1992; Zahier et al., 1992,
1993). A unique property of the SR proteins is that they
can be precipitated as a group in the presence of 20 mM
MgCi* (Zahier et al., 1992). Based on this property, a simpie two-step purification procedure was designed involv-
-
Figure 4. hSR Proteins Are Required
Formation of the dsxCC
wp*o
for the Tra- and T&-Dependent
Thesplicingsubstrate
is Dl. Thepositionsoftheprecursor
and splicing
product are indicated at the right of the autoradiogram.
Lane 1, unspliced substrate;
lane 2, incubation with nuclear extract in the presence of Tra and Tra2; lane 3, incubation
in the nuclear extract in the
presence of Tra, Tra2, and competitor
RNA 05; lanes 4-8, preincubation experiments,
with the experimental
schemes
used for each lane
illustrated below as in Figure 3. AS, 85%-90%
ammonium
sulfatesaturated
fraction of HeLa whole-cell extracts;
Mg (P), the pellet of
MgCk precipitation;
Mg (S/N), the supernatant
of MgCI, precipitation.
SDS-PAGE
analysis of the MgCI? precipitate
is shown at the right of
the autoradiogram.
The proteins are visualized by silver staining. Lane
M is the molecular weight standard.
The sizes of the marker proteins
are indicated at the left, Lane SR is the MgCl, precipitate.
The SR
proteins are labeled at the right.
Cell
108
AS
1
2
rSRp20
---a
3
4
5
rSF2/ASF
rSC35
cap55
/
6
7
8
9
IO 11 12
13 14
show comparable
activities in complementing
SlOO and
et al., 1992) SRpPO exhibits weak activities in these two
The absolute amount of each protein is not used here
activities for each protein preparation
are unknown.
For
of each protein.
d
r
a” +j* $
s
$9
-3 2@ $ Q
Figure
dsxCC
5. Complementation
of Tra and TraP in
Formation
by Individual SR Proteins
The splicing substrate
is Dl. Precursor
and
splicing product are indicated between the two
autoradiograms.
All lanes are preincubation
experiments
in the presence of Tra, Tra2, and
varying levels of SR proteins indicated above
each lane. Minus indicates
no SR proteins
added in the preincubation.
In the left-hand autoradiogram,
the three titration points for each
rSR protein contain 1 pi, 2 ul, and 4 pl of protein, respectively.
In the right-hand autoradio15 16 17 18 19 20
gram, 2 ul of rSC35 and 4 pl of each hSR protein are used. These amounts of SR proteins
in switching 5’ splice sites, except for SRpPO (data not shown). Analogous
to RBPI (Kim
assays. The amount of each protein given in microliters
serves only as a relative number.
for comparison
because the percentage
of active protein and the presence
of inhibitory
this reason, these data are not meant to give detailed quantitation
of the specific activities
Zahler et al., 1992). These results strongly suggest that
the SR proteins can complement Tra and Tra2 in dsxCC
formation.
Individual
SR Proteins Differ in Their Ability to
Promote dsxCC Formation
The MgCh precipitate contains at least six SR proteins
(Figure 4, lane SR; Zahler et al., 1992). To determine which
one or subset of the six SR proteins are responsible for
the complementing
activity, we generated recombinant
baculoviruses that express individual full-length SR proteins: SRp20, SFPIASF, SC%, and SRp55. The recombinant SR (rSR) proteins were purified with procedures analogous to those used for HeLa SR (hSR) proteins (see
Experimental Procedures). The other two SR proteins in
the MgCl* precipitate, SRp40 and SRp75, have not been
cloned (Zahler et al., 1992). Without recombinant versions
of these proteins, we purified them from the MgC& precipitate by preparative SDS-PAGE
(Zahler et al., 1992). We
then tested the activities of these proteins in dsxCC
formation.
As shown in Figure 5, rSC35 and rSRp55 efficiently complement Tra and Tra2 in the formation of the dsxCC (Figure
5, lanes 9-14). Similarly, hSRp40, hSRp55, and hSRp75
also complement Tra and Tra2 in dsxCC formation (Figure
5, lanes 18-20). In contrast, rSRp20, hSRp20, and rSF2/
ASF showed little or no activity in thecommitment
complex
assay (Figure 5, lanes 3-8 and 17). We also tested various
combinations of SR proteins in dsxCC formation and found
no synergy among them (data not shown).
Thus, individual SR proteins differ significantly in their
ability to function in dsxCC formation. SR proteins have
also been shown to function in two other assays: complementing SlOO for constitutive splicing activity and switching splice site usage (Ge et al., 1991; Krainer et al., 1991;
Fu et al., 1992; Mayeda et al., 1992; Zahler et al., 1992,
1993; Kim et al., 1992). In these two assays, SR proteins
also exhibit substratedependent
differences in activity
(Zahler et al., 1993). These observations and the distinct
behavior of SR proteins shown in this study suggest that
they may perform highly specific functions in regulated
splicing and that distinct sets of SR proteins may function
on different pre-mRNAs.
Tra and TraP Promote Complex Formation on the
dsx Repeat Element
The dsxCC may facilitate subsequent splicing by interacting with additional splicing components. For this reason, it is important to analyze complex formation on the
repeat element in the context of complete nuclear extract.
Gel filtration has been used to analyze the spliceosome
and its precursors (Abmayr et al., 1988; Reed et al., 1988;
Reed, 1990; Michaud and Reed, 1991; Bennett et al.,
1992a, 1992b), and we took a similar approach to study
complex formation on the repeat element.
The RNA we used for complex formation, D7, corresponds to a region of the female-specific exon that contains five copies of the repeat sequence (see Experimental
Procedures). As the negative control, we used an RNA
that has no repeat sequence, D8 (see Figure 2). After incubating “P-labeled RNAs in splicing reactions with or without Tra and Tra2, we loaded the reaction onto a Sephacryl
S-500 gel filtration column. The elution profile is generated
by measuring the radioactivity of the column fractions.
Three peaks of radioactivity were observed with the repeat-containing
RNA (Figure 8A). According to elution
volume, the peak eluting at fractions 38-45 represents
degraded RNA. The peak eluting at fractions 30-37 corresponds to H complex that results from the interaction
of heterogeneous
nuclear RNP (hnRNP) proteins with the
input RNA (Bennett et al., 1992a, 1992b). Both peaks are
present in elution profiles of a binding reaction with RNA
that does not contain the repeat sequence (Figure 8A).
The peak eluting at fractions 19-29 is unique to the RNA
containing the repeat sequence (Figure 8A). In addition,
the formation of this complex is significantly decreased in
the absence of Tra and Tra2 (Figure 8B). Thus, the formation of this complex is dependent on both the repeat element and Tra and Tra2. We call this complex the dsx repeat complex (dsxRC). The exact relationship between
the dsxRC and the dsxCC remains to be established. The
dsxCC is defined by its function in a commitment complex
ygitive
Regulation
of dsx Pre-mRNA
B
Splicing
Figure 6. Gel Filtration Analysis of the Complex Formed on the dsx Repeat Sequence
C
The graphs show the elution profiles of binding
reactions with RNAs plus or minus repeat sequence (A and C) and plus or minus Tra and
TraZ (B). These conditions plus the salt concentration used in binding reactions and in the column elution are indicated within each graph.
R and H indicate the dsxRC and the H complex,
respectively.
cpm
assay, it can form with purified Tra, Tra2, and SR proteins,
and it forms on a substrate containing both the repeat
sequence and a functional intron. In contrast, the dsxRC
has not been shown to be functional, it is formed in total
nuclear extract in the presence of Tra and Tra2, and the
RNA contains only the repeat sequence. As will be discussed later, the dsxRC may contain components in addition to Tra, Tra2, and SR proteins. Thus, the dsxCC may
be the core or precursor of the dsxRC.
In contrast with H complex, which is salt sensitive (Bennett et al., 1992b), the dsxRC can form and remains stable
in the presence of 250 mM KCI (Figure 6C). Under high
salt conditions, the amount of H complex is significantly
decreased, while the amount of the dsxRC increases
relative to the amount of complex formed under low salt
conditions. This observation suggests that under low salt
conditions H complex formation may compete with dsxRC
formation on the limiting amount of input RNA.
The dsxRC elutes primarily in the void volume of the
Sephacryl S500column
(Figure 6). Under the sameconditions, spliceosomes elute between the void volume and
H complex (Abmayr et al., 1988; Reed et al., 1988; Reed,
1990; Michaud and Reed, 1991; Bennett et al., 1992b).
The exclusion limit of Sephacryl S-500 resin is about
2 x 10’ daltons, so the dsxRC may be an aggregate of
monomeric complexes.
The dsxRC Contains Tra, Tra2, and SR Proteins
To analyze the composition of the dsxRC, we designed
an affinity purification method to separate it from the components in the nuclear extract (Figure 7). The method combines a modification of a previously described R17 coat
protein affinity method (Bardwell and Wickens, 1990) with
a biotin-avidin
affinity step (Grabowski and Sharp, 1986).
As shown in Figure 7, two copies of the binding site for the
bacterial phage R17 coat protein (Bardwell and Wickens,
1990) were inserted downstream of the dsx repeat element. Rather than covalently attaching purified R17 protein to agarose beads as previously described (Bardwell
and Wickens, 1990) we constructed a glutathione Stransferase-R17
fusion protein (GST-R17)
that could be
attached to glutathione beads and then released with glutathione (Smith and Johnson, 1988). The RNA containing
the dsx repeat sequences and the R17 coat protein-binding sites was synthesized in vitro in the presence of biotinUTP. The dsxRC formed in soluble reactions fails to bind
to glutathione-agarose
through GST-R17, presumably
owing to the aggregation of monomeric complexes. For
this reason, we first immobilized the RNA on glutathioneagarose. The dsxRC is then formed on the immobilized
RNA by mixing the resin with nuclear extract in the presence of Tra and Tra2 (Figure 7). The binding reaction was
carried out in 250 mM KCI to favor the formation of the
dsxRC relative to H complex.
After incubation at 30% for 1 hr, the unbound components in the reaction were washed away, and the proteinRNA complex was eluted by free glutathione (Figure 7).
The advantage of elution with glutathione is twofold. First,
the proteins bound nonspecifically to the resin are not released, further improving purification. Second, the gentle
elution condition does not disrupt the protein-RNA
complex, enabling further purification. When the complex was
eluted from the glutathione-agarose,
it was contaminated
dSX
m
r
------
4
R(i)
RI-I
Figure
7. Affinity
Purification
Scheme
I
for the dsxRC
The RNAs used in the purification are diagrammed
at the top. B represents biotin. The other symbols are explained in the diagram.
Cell
110
6
9
10
11
12
7
8
13
Figure 8. Analysis of the Complex
Tra, TraP, and Total SR Proteins
with Antibodies
Directed
against
The antibody used in each case is indicated at the top of each blot.
Lane M in each blot is the prestained
protein standard.
The sizes of
the marker proteins are indicated at the left of each blot. The last
lanes of each blot are purified proteins (Tra, Tra2, and SR proteins)
as positive controls.
The other lanes are complexes
formed under
conditions
indicated above each lane. The identities of the stained
proteins are indicated at the right of each blot.
with several glutathione-binding
proteins from the HeLa
cell nuclear extract. To remove these proteins, the protein-RNA complex was selected with avidin-agarose,
and
the proteins bound to the complex were then eluted with
buffers containing SDS (Figure 7).
After this second purification step, the protein composition of the eluted material is still complex when analyzed
on SDS-PAGE.
However, proteins unique to complex selected on repeat-containing
RNA could be detected, suggesting substantial enrichment for the dsxRC (data not
shown). As a first step in the analysis of the components in
the complex, we carried out Western blotting experiments
using antibodies against known splicing factors. When
Western blots of the affinity-purified
dsxRC were probed
with antibodies against Tra or Tra2, both proteins were
present in the complex assembled on RNA containing the
repeat sequence, but were not detected on RNA lacking
the repeat (Figure 8, lanes l-8).
We also used the monoclonal antibody (MAb), MAb104,
to detect SR proteins in the complex (Figure 8, lanes
9-l 3). MAb104 recognizes a phosphoepitope
present in
all SR proteins (Roth et al., 1991; Zahler et al., 1992).
This antibody also cross-reacts with Traand Tra2, possibly
owing to their phosphorylated
SR domains (Figure 8, lane
12). Western analysis with this antibody detected SRpSO
and SRp40 in the d.sxRC (Figure 8, lane 12). The staining
just below SRp40 is present in all the complexes and is
most likely due to the nonspecific staining of the GSTR17, because it is present in large amounts. SRp20 is
barely detectable in the dsxRC (Figure 8, lane 12). The
presence of SRp55 and SRp75 varies from very low (data
not shown) to nondetectable
(Figure 8, lane 12) depending on the batches of nuclear extracts used. The SR
proteins are detected only in the dsxRC formed on RNAs
containing the repeat sequence in the presence of Tra
and TraP (compare lanes 10 and 11 with lane 12 in Figure
8). Occasionally, a small amount of SRp30 could be detected in the complex in the absence of Tra and Tra2 (data
not shown).
The presence of SR proteins in the dsxRC further substantiates their roles in the regulation of dsx femalespecific splicing. SRp30 and SRp40 are the most abundant SR proteins in the complex. SRp30 is a mixture of
SC35 and SF2/ASF. Antibodies
against SF2/ASF detected only a small amount of SF2IASF in the dsxRC (data
not shown), suggesting that the majority of the SRp30 in
the complex is SC35. However, we have not been able
to prove this by staining with anti-SC35 antibody, since this
antibody recognizes a phosphoepitope
and cross-reacts
with other SR proteins (Fu et al., 1992). If the SRp30 in
the dsxRC is indeed accounted for by SC35, the presence
of SC35 and SRp40 would be consistent with the fact that
they efficiently complement Tra and Tr& in dsxCC formation. SRp55 and SRp75, although functioning in dsxCC
formation, are not consistently detected in the dsxRC. It
is possible that SRp55 and SRp75 interact with other factors in the nuclear extract. These interactions may modify
their specificities in protein-protein
or protein-RNA
interactions and prevent these two proteins from interacting
with the repeat element, with Tra and Tra2, or with both.
Small amounts of SF2/ASF and SRpPO are detected in
the dsxRC. Although these two proteins show no activity
in dsxCC formation, we cannot eliminate the possibility
that they function at later stages.
Analysis of the dsxRC by two-dimensional
gel electrophoresis revealed the presence of additional non-SR proteins (data not shown). The identities of these proteins are
currently being investigated.
Discussion
In this paper, we show that Tra, Tra2, and certain members
of the SR family of general splicing factors play an essential role in the assembly of the dsxCC. Moreover, we show
that these proteins stably associate with the dsxRC. Finally, we report that individual SR proteins differ significantly in their ability to cooperate with Tra and Tra2 in
promoting dsxCC formation. Thus, individual SR proteins
are not only able to function as essential splicing factors,
they can also interact with splicing regulators to control
alternative splicing.
Cooperative
Interactions
among Tra, Tra2, and SR
Proteins in dsxCC Formation
The cooperativity among Tra, Tra2, and SR proteins in
dsxCC formation is likely to involve both protein-RNA and
Positive
111
Regulation
of dsx Pre-mRNA
Splicing
protein-protein
interactions. Only one of these proteins,
Tra2, is capable of binding with high specificity to the dsx
repeat sequence in the absence of other factors (Hedley
and Maniatis, 1991) and this interaction requires the RRM
(H. Amrein, M.-L. Hedley, and T. M., unpublished data).
Tra can discriminate between oligonucleotides
containing
either the wild-type or mutant repeat sequence (Inoue et
al., 1992). When longer RNA fragments with higher sequence complexity are used in the binding reaction, the
binding specificity of Tra is variable, depending on the
assay used. In ultraviolet cross-linking experiments, Tra
binds RNA nonspecifically(Tian
and Maniatis, 1992) while
in filter binding assays Tra binds to the repeat element with
a low level of specificity (K. Wood and T. M., unpublished
data). However, Tra binds to the repeat element with a
high level of specificity in the presence of nuclear extracts
(Tian and Maniatis, 1992; this paper) or in the presence
of SR proteins (K. Wood and T. M., unpublished
data).
Similarly, specific recognition of the dsx repeat sequence
by SR proteins requires Tra and Tra2 (this paper; K. Wood
and T. M., unpublished
data). Thus, specific binding of
Tra and the SR proteins to the repeat element is likely to
involve interactions among Tra, Tra2, and SR proteins.
These observations and the fact that the dsx repeat element contains six copies of the repeat sequence suggest
that the initiation of dsxRC formation involves binding of
Tra2 to one or more of the repeats, followed by the recruitment of SR proteins and Tra to the complex, possibly
through interactions with repeat sequences not occupied
by Tra2 The RRMs of the SR proteins may be able to
recognize specifically one or more of the six repeat sequences when held in place by protein-protein
interactions with Tra, Tra2, or both. Once formed, this complex
may facilitate the assembly of other splicing factors on the
adjacent female-specific
3’ splice site.
Mechanisms
of Splicing Activation
The dsx female-specific
3’ splice site deviates from the
consensus sequence in that the pyrimidine tract immediately upstream of the AG dinucleotide
is interrupted by
purines (Burtis and Baker, 1989). Substitution of these
purines by pyrimidine leads to the constitutive activation
of this splice site in vivo (Hoshijima et al., 1991) and in
vitro(M. T. andT. M., unpublisheddata).
Thepurinesin
the
female-specific splice site most likely weaken the binding
of the splicing factor U2AF to the pyrimidine tract, which
is essential for the formation of the earliest ATP-dependent
prespliceosome
(A complex; Green, 1991). The Tra- and
T&-dependent
complex formed on the dsx repeat sequence could activate the female-specific
splice site by
stabilizing the weak interactions between U2AF with the
defective pyrimidine tract. The splicing factor UPAF is first
detected in E complex, the earliest known prespliceosome
complex formed during mammalian spliceosome assembly in vitro (Bennett et al., 1992b). E complex formation
occurs in the absence of ATP, and it commits pre-mRNA
to the splicing pathway (Michaud and Reed, 1991). E complex contains Ul snRNP. dsxCC formation may precede
E complex assembly since neither ATP nor Ul snRNP
is required in this process. The dsxCC may facilitate E
complex formation by interacting with U2AF and stabilizing its interaction with the defective pyrimidine tract. However, the recognition of the 3’ splice site by U2 snRNP
involves components in addition to UPAF (Kramer, 1988;
Fu and Maniatis, 1992b). These additional components
could equally be the targets of Tra and Tra2 activation.
Alternatively, Tra and Tra2 may interact with U2 snRNP
directly. The characterization
of the components in the
dsxRC should help distinguish among these possibilities.
A number of other examples of positive control of splice
site selection have been reported recently. The splicing
of the mouse immunoglobulin
u (Igfvl) pre-mRNA also involves a positive regulatory element (M2 element) (Watakabe et al., 1993). Analogous to the repeat element in dsx,
the M2 element is situated in the exon downstream of the
regulated 3’ splice site. Furthermore, the M2 element can
substitute for the repeat element in the dsxfemale-specific
exon and activate the female-specific
3’ splice site in a
Tra- and T&-independent
manner. Like the dsx repeat
element, M2 serves as a binding site for splicing components that may include Ul snRNP. The M2 element consists of a purine-rich sequence, and similar sequence motifs have been found in the exons of other pre-mRNAs.
The dsx repeat sequence shares no homology with the
purine-rich exon elements, consistent with its nature as
a specialized regulatory element. The role of exon sequences in splicing has been recognized in early studies
(Reed and Maniatis, 1988); however, their exact function
remains unclear. The present observations with the dsx
repeat element and the IgM M2 element suggest that exon
sequences may contain binding sites for regulatory complexes.
Another case of positive regulation is the neuronalspecific splicing of c-sfc (Black, 1992). In contrast with the
dsx repeat element and IgM M2 elements that are in the
exon, the c-src regulatory element is situated in the intron
downstream of the regulated splice sites. Although different in location, the C-SC element also serves as a binding
site for splicing activators, the identities of which are unknown at present. Finally, in exon definition, factors binding to 5’ and 3’ splice sites communicate across the exon
(Robberson et al., 1990) and the 3’splice sitesof terminal
exons can interact with polyadenylation
factors (Niwa and
Berget, 1991; Niwa et al., 1992). In the most recent example involving the alternative splicing of exon 4 in preprotachykinin pre-mRNA, the binding of Ul snRNP to the 5’
splice site at the 3’ end of exon 4 facilitates the binding
of U2AF to the 3’ splice site at the 5’ end of this exon
through exon-bridged
interactions (Hoffmann and Grabowski, 1992).
All these cases of splicing activation involve positive
regulatory complexes. The elements differ in their position: the dsx repeat element and IgM M2 element are located in exons downstream of the affected 3’ splice site,
the c-srcelement maps to an intron, and the preprotachykinin element is positioned at the intron-exon
boundary.
However, these elementsshare
one thing in common: they
are all located adjacent to and downstream of the regulated splice sites. This arrangement is reminiscent of tran-
Cell
112
scription activation by enhancers. Thus, these elements
may function as splicing enhancers.
In contrast with the positive regulation discussed above,
regulatory proteins can also antagonize the function of
general splicing factors. Sex lethal (Sxl) regulates the alternative splicing of tra pre-mRNA by binding to the pyrimidine tract of the regulated splice site and precluding the
interaction of U2AF with this splicing signal (Valcarcel et
al., 1993). The germline-specific
splicing of P element premRNA is controlled by inhibitory complexes assembled
on pseudod’splice
sites that interfere with the recognition
of the adjacent authentic 5’ splice site (Siebel and Rio,
1990; Siebel et al., 1992). Su(w”) protein represses the
splicing of both its own primary transcript and the whiteapricot
pre-mRNA (Singham et al., 1988). Given the various interactions between splicing regulators and general
splicing components, studies in this and other alternative
splicing systems should contribute to the understanding
of both regulated and constitutive splicing.
Expsrlmental
Procedures
In Vitro Splicing
Reactions
In vitro splicing reactions
were carried out as previously
described
(Tian and Maniatis, 1992). In commitmentcomplexassays,
thesplicing
substrate was preincubated
with the reagents indicated plus the basic
components
in the splicing reaction (ATP, creatine phosphate,
MgCI,,
polyvinyl alcohol) unless otherwise
specified.
After preincubation
at
30°C for 1 hr, the reagents indicated at the second addition point were
added, and the incubation
was continued
for another 2 hr.
Proteins
and Antibodles
The antibody for Tra is a mouse polyclonal antibody generated against
purified recombinant
Tra. The antibody for Tra2 is a mouse MAb generated against purified recombinant
Tra2. The antibody for SF2/ASF is
a gift from Drs. A. Krainer and A. Mayeda. MAb104 is obtained from
the hybridoma
provided by Dr. M. Roth.
The cDNAs for SRpPO and SRp55 were amplified from HeLa cell
and Drosophila
embryo cDNAs. To facilitate detection,
the 5’ ends of
the cDNAs of SRpPO and SRp55 were fused to a 10 amino acid myc
tag. The SF2/ASF cDNA was not modified. The cDNAs containing the
complete coding regions of SRpPO, SRp55, and SFP/ASF were cloned
into the baculovirus
expression
vector pVL941 (PharMingen),
and the
recombinant
baculoviruses
were generated according
to the protocols
of the manufacturer
(PharMingen).
The recombinant
virus for SC35
was described previously (Fu and Maniatis, 1992a). For protein production, Sf9 cells were infected with individual virus. All the procedures
were carried out at 4“C. After 72 hr of infection, the cells were spun
down at 2000 rpm (TJ-6 rotor) for IO min and washed once with phosphate-buffered
saline. The cells from a 100 ml culture were lysed in
20 ml of buffer A (20 mM Tris-HCI
[pH 7.51, 100 mM KCI, 0.2 mM
EDTA, 1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol
[OTT])
by sonication.
The cell debris was spun down at 8000 rpm (HB-4 rotor)
for 20 min and the supernatant
saved. The debris was reextracted
with 20 ml of buffer B (20 mM Tris-HCI
[pH 7.51.600 mM ammonium
sulfate, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 0.5%
p-mercaptol
ethanol) for 30 min. The mixture was spun at 6900 rpm
(HE-4 rotor) for 20 min. The supernatant
was pooled with that from
the first spinning.
Ammonium
sulfate was added to the supernatant
to 40% saturation. After stirring for 2 hr, the mixture was spun at 10,000
rpm (HB-4 rotor) for 30 min. The supernatant
was loaded onto a 2 ml
Phenyl-Sepharose
(Pharmacia)
column equilibrated
in buffer C (20
mM Tris-HCI
]pH 7.51, 0.2 mM EDTA, 1 mM DTT) containing
1.8 M
ammonium sulfate. The elutions were carried out in steps using buffer
C containing 1.3 M. 1 .O M. and 0.5 M ammonium sulfate, respectively.
rSR2/ASF
and rSC35 were eluted in the 1.3 M and 1 .O M fractions,
and rSRp20 and rSRp55 were eluted in the 1 .O M and 0.5 M fractions.
The fractions
containing
SR proteins were dialyzed against BClOO
(20 mM Tris-HCI
[pH 751,100 mM KCI, 0.2 mM EDTA, 0.5 mM Dll).
The SR proteins were purified from the dialyzed fractions
by MgClz
precipitation
(Zahler et al., 1992). As positive controls for the activities
of the rSR proteins,
they all function
in complementing
SlOO and
switching 5’ splice site assays, although rSRp20 displays weak activities in both assays, analogous to the RNA-binding
protein RBPl (Kim
et al., 1992).
GST-R17
was produced
by Escherichia
coli transformed
with
pGST-R17.
pGST-RI
7 was constructed
by cloning the RI 7 coding
sequence
(Gott et al., 1991) into GST expression
vector (pGEM-A)
(Smith and Johnson,
1988). The transformed
E. coli (HBlOl) (800 ml)
was grown to OD,
= 0.4-0.5,
and isopropyl
6-D-thiogalactopyranoside was added to a final concentration
of 0.125 mM. The cells
were induced for 3 hr. The cells were spun down at 4000 rpm (J-6
rotor) and washed with 20 ml of buffer I (50 mM Tris-HCI
[pH 6.01,
25% sucrose,
10 mM EDTA). The pellet was resuspended
in 20 ml
of buffer I. Four milliliters of 20 mglml lysozyme in buffer I were added
to the cells, and the digest was incubated
on ice for 1 hr. The cells
were spun down at 8000 rpm (HE-4 rotor) for 10 min and then lysed
by sonication
in 20 ml of buffer II (10 mM Tris-HCI
[pH 7.51, 1 mM
EDTA, 1 mM DlT, 1 mM phenylmethylsulfonyl
fluoride, 1 mM benzimidine). The cells were lysed by sonication.
KCI and Triton X-100 were
added to 100 mM and 0.1% final concentrations,
respectively.
The
debris was pelleted by spinning at 8000 rpm (HB-4 rotor) for 30 min.
The supernatant
was loaded onto a 2 ml glutathione-agarose
column
(Sigma) equilibrated
with buffer Ill (20 mM Tris-HCI
[pH 7.51, 100 mM
KCI, 0.2 mM EDTA, 1 mM DTT). GST-R17
was eluted with buffer Ill
containing
5 mM glutathione.
The eluted GST-R17
was dialyzed
against buffer III for 24 hr.
Gel Flltratlon
Analysis
Splicing reactions were scaled up to 200 pl for gel filtration analysis.
Polyvinyl alcohol was left out of the reaction, and KCI concentration
was as specified. The reactions were incubated at 30°C for 1 hr and
then loaded onto a Sephacryl S-500 (Pharmacia)
column (1.5 cm x
50 cm). The column was equilibrated
and run in 20 mM Tris-HCI
(pH
7.5) 60 or 250 mM KCI (as specified),
0.2 mM EDTA, 0.1% Triton
X-i 00 at a speed of 7 mllhr. Fractions
(1.8 ml) were collected,
and
0.2 ml samples of each fraction were counted by Cherenkov
counting.
RNA8
Dl , D2, D3, D4, D5, and D6 were as described
previously
(Ran and
Maniatis,
1992). D7 was transcribed
from T3F260 linearized
with
Hindlll. T3F260 was constructed
by cloning into Bluescript SK(+) vector a 260 bp Fspl fragment of the dsx female-specific
exon. The R(+)
RNA used in the purification
of the dsxRC is transcribed
from R(S)R17 linearized with Bglll. R(S)-R17
was constructed
by ligating a 390
bp Mlul-Aflll fragment from the female-specific
exon to two copies of
the R17-binding
site (Bardwell and Wickens,
1990). The fusion construct is contained
in the SP73 vector (Promega).
The R(-) RNA is
transcribed
from Afl-RI7
linearized
with Bglll. AR-RI7
was constructed by ligating a 380 bp Aflll fragment from the female-specific
exon to two copies of the R17-binding
site (Bardwell
and Wickens,
1990).
Afflnlty
Purlflcatlon
of the dsxRC
RNA (4 ug) was incubated
with 24 ug of GST-R17
in TMK buffer
(0.1 M Tris-HCl
[PH 8.01, 10 mM MgC12, 0.1 M KCI) in 25OC for 50
min. A 1:l slurry (1 ml) of glutathione-agarose
(equilibrated
in TMK
buffer) was added to the reaction. Triton X-100 was added to 0.01%
final concentration.
The mixture was rocked at 4OC for 1 hr. The resin
was washed four times with 4 ml each of S buffer (20 mM Tris-HCI
[pH 7.51, 250 mM KCI, 3 mM MgCI,, 0.01% Triton X-100). The resin
was mixed with a 2 ml splicing reaction. The composition
of the splicing
reaction is the same as that in normal splicing except that the KCI
concentration
is 250 mM and the polyvinyl alcohol is omitted. The
reaction was rocked at 30°C for 1 hr. The resin was washed eight
times with 4 ml each of ST buffer (S buffer plus 0.1% Triton X-100).
After washing, 2.5 ml of ST buffer containing
50 mM glutathione was
added to the resin, and the mixture was rocked at 4OC for 30 min.
The resin was spun down, and the supernatant
was saved. The elution
was repeated once more. The two elutions were pooled. A I:1 slurry
(200 ul) of avidin-agarose
(equilibrated
in S250: 20 mM Tris-HCI
[pH
7.51, 250 mM KCI, 0.2 mM EDTA, 0.1% Triton X-100) was added to
y$ive
Regulation
of dsx Pre-mRNA
Splicing
the eluant. and the mixture was rocked at 4OC overnight.
The resin
was washed four times with 10 ml each of S250. SDS buffer (0.8 ml)
(20 mM Tris-HCI
[pH 7.51, 2% SDS, 5% p-mercaptol
ethanol) was
added to the resin. The mixture was incubated at 85OC for 10 min.
The resin was spun down and the supernatant
saved. The elution was
repeated once more. The supernatants
were pooled, and the proteins
were precipitated
with 4 vol of acetone. The pellet was resuspended
in SDS sample buffer and run on SDS-PAGE.
Western
analysis was
done according
to standard procedures
(Promega).
Acknowledgments
We thank Adrian Krainer and Akila Mayeda for the SlOO extract and
anti-SF2/ASF
antibody;
Robin Reed for the 5’ D-16X plasmid; Mark
Roth for the hybridoma producing
MAb104; Marvin Wickens
and Olke
Uhlenbeck
for plasmids used in the R17 procedure;
and Hubert Amrein, James Bruzik, Xiang-Dong
Fu, Mary-Lynne
Hedley, Edward C.
Hsiao, Robin Reed, Tom Schaal, Kristen Wood, and Jane Wu for discussions
and comments
on the manuscript.This
work is supported
by
a grant from the National Institutes of Health to T. M
Received
April 6, 1993;
revised
May
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