Critical protein-protein interactions among
pre-mRNA splicing factors determine splicing efficiency and
specificity. The serine/arginine proteins, a family of factors
characterized by the presence of an RNA recognition motif and an
arginine/serine domain, are essential for constitutive splicing and
required for some alternative splicing decisions. ASF/SF2, SC35, and
other members of the serine/arginine family, interact with the 70k
protein of the U1 small nuclear ribonucleoprotein. The binding of this
protein with ASF/SF2 is thought to enhance recognition of the 5' splice
site of pre-mRNAs by the U1 small nuclear ribonucleoprotein. It has
been clearly documented that the arginine/serine domain of ASF/SF2 is
responsible for binding to the U1 70k protein. In this manuscript we
characterize the segment in the human U1 70k protein that is both
necessary and sufficient for ASF/SF2 binding. A domain within this
segment, which begins with Arg240 and ends with
Asp270, was shown to bind specifically to the
arginine/serine domain of ASF/SF2 using a yeast two-hybrid system and a
far Western assay. Mutational analysis of this segment suggested that
several arginines are critical for the interaction with ASF/SF2 and for
phosphorylation by SRPK1. Inspection of the sequence of the
Arg248 to Asp270 region suggested this as an
arginine/serine-like domain in U1 70k protein, and the data presented
in this manuscript strongly support this view. Inspection of the human
U1 70k protein sequence, comparison with homologues in other animal
species, and mutational analysis indicated the importance of the
sequence Arg-Arg-Arg-Ser-Arg-Ser-Arg-Asp, which is found repeated twice
in the region from Arg248 to Asp270 in the
human protein.
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INTRODUCTION |
U1 snRNP, the first of the spliceosomal
snRNPs1 to assemble with the
pre-mRNA (1-4), employs both RNA and protein components to
recognize the 5' splice site (5). Base pairing between the 5' end of U1
snRNA and the 5' splice site of the pre-mRNA is a critical early
interaction in formation of the spliceosome (6). Additionally,
recognition of the 5' splice site by the U1 snRNP requires U1-specific
proteins, including the U1 C protein (7, 8) and possibly U1 70k protein
(8, 9). U1 70k contains an RNA-binding domain of the RNA recognition
motif type, which binds stem-loop A of U1 snRNA (10). U1 70k also has
two arginine-rich regions punctuated by serine or acidic residues
(10-14). Although neither arginine-rich region contains extended RS
dipeptide repeats, such as found in ASF/SF2 and SC35, the two regions
have been designated RS1 and RS2. The C-terminal half of U1 70k, which
contains the RS1 and RS2 regions, has been implicated in
protein-protein interaction with SR proteins that mediate 5' splice
site recognition (9).
A requirement for U1 70k in pre-mRNA splicing has been demonstrated
genetically in Saccharomyces cerevisiae (yeast) for some but
not for all introns (15). Genetic analysis has not been illuminating on
the function of regions of this protein in yeast. Neither the RNA
recognition motif nor the C-terminal region of the protein is
absolutely required for function in vivo (15). Yeast U1 70k
does not have easily recognizable arginine/serine-rich domains.
The superfamily of RS domain-containing splicing factors is composed of
two subfamilies of proteins (reviewed in Ref. 16). The first is the
family of SR proteins that includes SRp20 (17), ASF/SF2, also known as
SRp30a (18-20), SC35, also known as SRp30b (21, 22), Srp30c (23),
SRp40 (24), SRp55/B52 (17), SRp75 (25), 9G8 (26), and NP13, a yeast SR
protein (14). Most SR proteins share two structural motifs: an RNA
recognition motif (10) and an RS domain (17). The RS domain is
essential for the in vivo function of the Drosophila
melanogaster splicing regulator Suppressor of white
apricot protein (SWAP) (27) and for the in vitro
constitutive splicing activity of ASF/SF2 (28, 29). The second
subfamily is composed of SR protein-related factors and includes, among
others, the splicing regulators Tra (30) and Tra-2 (31, 32). U1 70k
protein has also been included as a member of this second subfamily
(16).
Individual SR proteins function with varying efficiency in the splicing
of different pre-mRNAs (26, 33, 34). Moreover, ASF/SF2 has been
shown to be essential for viability in a chicken B cell line,
indicating a unique role in vivo for this SR protein (35).
Nevertheless, in other assays SR proteins can substitute for each
other, suggesting that SR proteins have partially redundant functions
in splicing in vitro (17, 26, 36, 37). SR proteins interact
with each other and with other splicing factors during the formation of
spliceosome (9, 38, 39). ASF/SF2 and SC35 interact with U1 70k (9, 38),
and the ASF/SF2 RS domain is required for this interaction (9). The
binding of ASF/SF2 and U1-70k is thought to stabilize the association
of the 5' splice site with the U1 snRNP. Similar interactions may
explain the SR protein-mediated enhancement of U2 snRNP binding to
pre-mRNAs and U4·6,5 tri-snRNP to assembling spliceosomes (40,
41). Whereas the 70k equivalent in U2 snRNP has not been identified, the U5 snRNP-associated p27 contains a putative RS domain and is likely
involved in binding SR proteins (42). Thus, the interaction between U1
70k and SR proteins may be a model for a broader set of interactions
between snRNP proteins and SR proteins.
We had previously shown that the C-terminal half of U1 70k was required
for binding ASF/SF2 in a far Western assay (9). The determinants of
this binding were investigated, and here we show that the RS1 region in
70k is necessary and sufficient for binding ASF/SF2. By extension, we
point out that the RS2 region is likely to have other roles in the
function of U1 70k. We dissected the RS1 region and show that the
segment encoded by amino acids 248-270, which we call RS1nc, was
sufficient to replace the RS1 region. Moreover, we present data that
indicate the importance of arginine residues within a sequence repeated
in the segment Arg248-Asp270 in RS1. Several
of these arginines are also critical for the phophorylation of the
segment by SRPK1. Thus, the Arg248-Asp270
segment in the U1 70k protein is equivalent to an RS domain.
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MATERIALS AND METHODS |
Constructs and Proteins--
Segments of a ASF/SF2 cDNA were
subcloned into the plasmid pGBT9 (CLONTECH)
encoding the GAL4 DNA-binding domain using the EcoRI and
BamHI sites. Segments of a U1 70K cDNA were subcloned into the plasmid pGAD424 (CLONTECH), which encodes
the GAL4 transcription activation domain (GAL4AD), between the
EcoRI and BglII sites. The constructs ASF,
ASF-RS, ASF-RSn, and ASF-RSc encode for the full length, the RS domain,
the N-terminal half of the RS domain of ASF/SF2, and the C-terminal
half of the RS domain of ASF/SF2, respectively. ASF
RS lacks an RS
domain. Constructs U1 70K, 70K-RS1, and 70K-RS2 encode the full-length,
the RS1 domain, and the RS2 domain of U1 70K protein, respectively.
271-437,
249-437, and
231-437 represent U1 70K constructs
with deletions of amino acids 271-437, 249-437, and 231-437,
respectively. RS1n and RS1c constructs encode the N-terminal segment or
C-terminal segment of the RS1 region, and RS1nn and RSnc contain the
N-terminal segment or the C-terminal segment of SR1n. RS1nc-mut is a
derivative of RS1nc, in which Arg260 was mutated to lysine
(this mutant will be designated m6). 70K-
RS1nc was constructed by
deleting the region encoding amino acids 240-300 of U1 70K using
SmaI and ApaI restriction endonucleases (New
England Inc.). Subsequently, the region encoding amino acids 271-300
was cloned back as a polymerase chain reaction product. The variants of
271-437 (m1 to m5) were made by cloning annealed oligonucleotides so as to extend the open reading frame in construct
249-437. DNA
fragments encoding either 70k-SR1nc or mutant SR1nc segments m1, m3,
and m7 (see Fig. 4A) were subcloned into the pGEX-2TK vector
(Amersham Pharmacia Biotech) using the BamHI and
EcoRI sites. These plasmids were used to overexpress GST
fusion proteins as described by Sune and Garcia-Blanco (43).
Oligonucleotides encoding a (SR)5 peptide, SRSRSRSRSR, were
subcloned into pBGT9 vector in the EcoRI site. Recombinant
ASF/SF2 were made as described previously (20). Baculovirus-derived
SRPK1 was a kind gift from X.-D. Fu (University of California at San
Diego). The vector GST-SRPK1 was a gift from J. Fleckner (Aarhus,
Denmark). GST-SRPK1 fusion protein was prepared from Escherichia
coli as described in Ref. 43.
Yeast Two-hybrid Interaction Experiments--
The two-hybrid
interaction experiments were performed according to Fields and Song
(44). The strain SFY526 (Mata, ura3-52, his3-200, ade2-101,
lys2-801, trp1-901, leu2-3, 112, canr, gal4-542, gal80-538,
URA3::GAL1-lacZ) was used as the host yeast strain for all
the experiments. To assay the pairwise interactions, segments of
cDNAs encoding the U1 70k protein were introduced into vector
pGAD424 and that of cDNA encoding ASF/SF2 protein and
(SR)5 peptide into the vector pGBT9 and in some cases also in pGAD424. The liquid
-galactosidase assay was carried out by using
the yeast extracts according to Fridell et al. (45) and was
normalized using the A600 of yeast cultures.
Phosphorylation of GST-SR1nc Fusion Proteins--
The
recombinant GST-SR1nc fusion protein and the mutant GST-M1, GST-M3, and
GST-M7 fusion proteins, which were encoded by plasmids with RS1nc
encoding fragments designated m1, m3, and m7, respectively (see
sequence in Fig. 4A), were purified on glutathione-agarose beads as described (43). Phosphorylation reactions were carried out in
50 µl and contained 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM dithiothreitol, 1 µCi of [
-32P]ATP, and either 5 ng/µl recombinant
SRPK1 or 10 µl of HeLa S100 extract, added to 20 µl of packed
glutathione-agarose beads for 90 min at 30 °C. The beads were washed
with 100-150 packed bead volumes, resuspended in SDS-PAGE loading
buffer, and loaded onto 12.5% SDS-PAGE gels. The phosphorylated
proteins were visualized by autoradiography.
Western and Far Western Blot Analyses--
Yeast lysates (equal
amount for each lane) were separated on 12.5% SDS-PAGE gel and
transferred to Immobilon-P (Millipore Inc.) membrane for Western or
nitrocellulose (GeneScreen Inc.) for far Western blot analyses using a
25 mM Tris and 192 mM glycine/20% methanol
transfer buffer. Monoclonal antibody of U1 70K (46) and monoclonal
antibody mAb104 (47) were used for Western blot analyses. A secondary
anti-mouse Ig antibody linked to horseradish peroxidase was used in ECL
Western assay (American Life Science) to detect the monoclonal
antibodies and was used as described by the manufacturer. In far
Western assays recombinant ASF/SF2 protein was labeled with using
[
-32P]ATP and SRPK1. Labeling used 200 ng/µl
ASF/SF2, 15 ng/µl SRPK1, 50 mM Tris (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol, and 1 mM ATP. The labeled ASF/SF2 protein was subsequently
purified through nickel columns (Qiagen) as described in Ref. 20.
Immediately before use, filters for far Western blots were incubated in
modified buffer D containing 6 M guanidine HCl for 30 min
at room temperature (48). Proteins were renatured by dilution (1:1) in
buffer D for six cycles (10 min each). Filters were then rinsed twice
in buffer D and blocked in buffer D containing 5% nonfat dry milk and
0.1% Tween 20 for 1 h. Subsequently, the filters were incubated with labeled ASF/SF2 in the binding buffer containing 13 mM
HEPES (pH 7.9), 28 mM
(NH4)2SO4, 33 mM KCl,
3.3 mM MgCl2, 0.13 mM EDTA, 2%
(w/v) nonfat dry milk, 0.1% Tween 20, 0.5 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride
for 12 h at 40 °C. Blots were washed three times for 20 min in
binding buffer without dry milk, dried, and subjected to
autoradiography.
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RESULTS |
Sequences within the RS1 Region of U1 70k Protein Are Necessary and
Sufficient for Binding ASF/SF2--
The interaction between ASF/SF2
and U1 70k protein has been shown to depend on the RS domain of ASF/SF2
(9, 38). Moreover, the C-terminal half of 70k protein, which contains
the RS1 and RS2 regions (Fig.
1A), was also shown to be
required (9). To characterize the sequences in U1 70k that are
necessary and sufficient to bind ASF/SF2, we tested for this binding
in vivo using the two-hybrid system of Fields and Song (44).
Chimeric proteins, with segments of U1 70k protein fused to the
transcriptional activation domain of GAL4 (GAL4AD), were co-expressed
with chimeric proteins containing the open reading frame of ASF/SF2
fused to the DNA-binding domain of GAL4. The relative strength of
protein-protein interactions were determined by quantifying
-galactosidase activity in yeast extracts.

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Fig. 1.
The RS1 region of U1 70k contains sequences
that are required for ASF/SF2 binding. A, the complete
amino acid sequence of human U1 70k protein according to Query et
al. (10). B, diagram showing the regions of the human
U1 70k protein (numbering of amino acid residues is based on Query
et al. (10) fused to the GAL4AD and tested for ASF/SF2
binding in the yeast two-hybrid system. C, the relative
strength of interactions between U1 70k or deletion mutants and ASF/SF2
was determined by measuring -galactosidase activity. Quantitative
liquid -galactosidase assays were performed on at least three
independent experiments for each combination, and the mean values are
shown in the bar graph. The level of background -galactosidase
activity obtained with the negative control, which was obtained by
cotransfection of empty vectors, was set at 1.0. The interactions of
ASF with U1 70k, 271-437, 249-437, and 231-437 were
measured at 46.9 ± 3.5, 25.6 ± 2.4, 0.9 ± 0.3, and
0.9 ± 0.2, respectively. These values represent the mean
values ± S.D.
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The requirement for the RS1 and RS2 regions of the U1 70k in ASF/SF2
binding was investigated by sequentially deleting C-terminal segments
of U1 70k (Fig. 1B). Deletion of the RS2 region and the C-terminal region of the RS1 region (
271-437) resulted in a protein that retained significant binding to ASF/SF2 (Fig. 1C). This
deletion removes the RS2 region, which is rich in arginines and in
acidic residues (Fig. 1A) (10). It also removes a section of
the RS1 region that contains four RS dipeptides (see amino acids
276-296 in Fig. 1A) but is devoid of runs of these RS
dipeptides. Further deletion to amino acid 248 (
249-437), which
eliminates two runs of RS dipeptides, completely abolished binding and,
as expected, so did deletion to amino acid 230 (
231-437) (Fig.
1C). This experiment indicated that amino acids 248-270
were important for ASF/SF2 binding and suggested a modest contribution
by elements within the region from amino acid 271 to the C
terminus.
To further characterize the sequence requirements for ASF/SF2 binding,
we constructed GAL4AD fusion proteins with short segments of U1 70k
(Fig. 2A). The RS1 and the RS2
regions were tested, and only the former was capable of binding ASF/SF2
(Fig. 2B). Although the binding of the isolated RS1 was
always lower (~70%) than the binding of the full-length U1 70k, this
difference was not highly significant (Table
I). The RS1 region was divided into two
subregions, RS1n and RS1c (Fig. 2A), each of which contained at least one Arg-Ser dipeptide. RS1n, which spans amino acids 230-270,
bound ASF/SF2 almost as well as RS1, whereas the RS1c subregion, which
encompasses residues 271-309, did not (Fig. 2B). RS1n was
divided into two segments: RS1nn, which did not contain Arg-Ser
repeats, and RS1nc, which contained two RSRSR pentapeptides. Not
surprisingly, the RS1nn segment did not bind ASF/SF2, whereas the RS1nc
did (Fig. 2B). This 23-amino acid-long segment, which spans
residues 248-270, bound ASF/SF2 almost as well as did the RS1 region,
and about half as well as full-length U1 70k protein (Fig.
2B). Thus, the segment between amino acids 248 and 270 of the U1 70k protein was sufficient for ASF/SF2 binding.

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Fig. 2.
Sequences within the RS1 region of U1 70k are
sufficient for ASF/SF2 binding. A, diagram showing the
regions of the human U1 70k protein (numbering of amino acid residues
is based on Query et al. (10)) fused to the GAL4AD and
tested for ASF/SF2 binding in the yeast two-hybrid system.
B, the relative strength of interactions between segments of
the U1 70k and ASF/SF2 was determined by measuring -galactosidase
activity. Quantitative liquid -galactosidase assays were performed
in at least three independent experiments for each combination, and the
mean values are shown in the bar graph. The level of background
-galactosidase activity obtained with the negative control was set
at 1.0. These data are expressed numerically as the mean ± S.D.
in the first column of Table I.
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Table I
Interactions between regions of U1 70k protein and ASF/SF2
A quantitative liquid -galactosidase assay was performed on at least
three independent experiments for each combination (45). The yeast
extract was normalized by A600 of the yeast
cultures. The numbers shown represent the fold activation over the
negative control obtained when plasmids pGBT9 and pGAD424, the parental
plasmids, were tested for interactions and are expressed as the
means ± S.D. All ASF/SF2 segments were cloned into pGBT9, whereas
U1 70k segments were cloned in pGAD424.
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To determine whether or not the RS1 region was absolutely necessary for
binding ASF/SF2 we constructed a plasmid encoding a GAL4AD fusion
protein that contained the full length of U1 70k with a precise
deletion of amino acids 240-270 (
RS1nc in Fig. 3A). It should be noted that
the borders of this deletion are not equivalent to those of the RS1nc
fragment; the deletion extends to eight residues N-terminal of the
RS1nc fragment (compare Fig. 2A with Fig. 3A).
Full-length U1 70k and
RS1nc were tested, and the deletion of amino
acids 240-270 diminished binding ASF/SF2 by more than 4-fold (Fig.
3B). It is important to note that in these experiments the
negative control is arbitrarily assigned a value of 1.0 (not shown),
which implied that the
RS1nc was still able to weakly bind ASF/SF2.
These data strongly suggested that the segment from 240-270 was
necessary for efficient ASF/SF2 binding.

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Fig. 3.
The RS1nc segment of U1 70k is necessary and
sufficient for ASF/SF2 binding. A, diagram
showing the regions of the human U1 70k protein (numbering of amino
acid residues is based on Query et al. (10)) fused to the
GAL4AD and tested for ASF/SF2 binding in the yeast two-hybrid system.
B, the relative strength of interactions between segments of
the U1 70k and ASF/SF2 was determined by measuring -galactosidase
activity. Quantitative liquid -galactosidase assay were performed on
at least three independent experiments for each combination, and the
mean values are shown in the bar graph. The level of background
-galactosidase activity obtained with the negative control was set
at 1.0.
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The RSRSR Pentapeptides in U1 70k Are Important for ASF/SF2
Binding--
The segment of U1 70k spanning amino acids 248-270,
which we refer to as RS1nc (Fig. 2) and which was sufficient for
ASF/SF2 binding, contains two RSRSR pentapeptides. We investigated the role of four arginines, Arg258, Arg260,
Arg267, and Arg269, that occupy the positions
underlined in the RSRSR pentapeptides (Figs. 1 and
4A). Arg260 was
changed to Lys260 in the context of an isolated RS1nc
segment in construct RS1nc-mut (Fig. 3A). This change
significantly diminished ASF/SF2 binding in the two-hybrid system
compared with the RS1nc (Fig. 3B). In this experiment the
RS1nc-mut had the same low level of binding as the full-length protein
with a deletion of amino acids 240-270 (
RS1nc).

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Fig. 4.
Important arginine residues in the RS1nc
segment of U1 70k. A, diagram showing residues 251-270
of the human U1 70k protein and variants of this sequence, which were
made in the context of the 271-437 protein (see Fig.
1B). These were made as fusions with GAL4AD and tested for
ASF/SF2 binding in the yeast two-hybrid system. B, the
relative strength of interactions between 271-437 or variants and
ASF/SF2 was determined by measuring -galactosidase activity.
Quantitative liquid -galactosidase assay were performed on at least
three independent experiments for each combination, and the mean values
are shown in the bar graph. The level of background -galactosidase
activity obtained with the negative control was set at 1.0. The
values ± S.D. were: for 271-437, 9.3 ± 1.8; for m1,
1.8 ± 0.2; for m2, 2.5 ± 0.5; for m3, 1.8 ± 0.0; for
m4, 2.4 ± 0.2; for m5, 1.4 ± 0.3. C, the
GST-SR1nc and GST-SR1nc mutants M1, M3, and M7 (see Fig. 4A) were
purified and phosphorylated as described under "Methods and
Materials." The phosphorylated proteins were separated in a 12.5%
SDS-PAGE gels and visualized by autoradiography. The top
panel shows the extent of phosphorylation of GST protein
(lane 1), GST-SR1nc (WT, lane 2), or
GST-Sr1nc mutants M1, M3, and M7 (lanes 3-5). The concentrations of the different GST fusion proteins in the
phosphorylation reactions were very similar, as can be seen by
Coomassie Blue staining of a gel run in parallel (bottom
panel).
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We constructed a library of variants of the clone
271-437 (Fig.
1B) where the aforementioned four amino acid positions could be randomly occupied by arginine or lysine. Screening for independent clones did not yield all possible mutants; nevertheless, the wild type
(
271-437) and five mutants were obtained and analyzed. Three mutants (m2, m3, and m5) had double Arg
Lys substitutions that changed both RSRSR pentapeptides. All of these reduced ASF binding to
~25% of the
271-437 (Fig. 4B). This level of binding
was clearly above that obtained with
249-437, a mutant missing the
segment with the two pentapeptides (Fig. 1B), suggesting
that the Arg
Lys mutations crippled but did not destroy the binding
domain. Mutations in the C-terminal RSRSR pentapeptide (m1 and m4) had very similar effect (Fig. 4B). One of these, m1, was the
replacement of a single arginine, Arg269, for
Lys269 (Fig. 4B), indicating that
Arg269 was critical for binding. Given these results and
those obtained with RS1nc-mut, it is likely that both pentapeptides
play equivalent and cooperative roles and that mutation of either RSRSR
pentapeptide would be detrimental.
To confirm that these mutant GAL4AD proteins are expressed to
equivalent levels, we measured their levels using a monoclonal antibody
to U1 70k (46). The levels of
271-437 (wild type) and the mutants
m1 to m5 were approximately the same, as were the levels of
249-437
and GAL4AD-U1 70k (data not shown). The levels of the ASF/SF2 fusion
protein were also determined using the monoclonal antibody mAb104 (47).
The levels were somewhat varied; however, if anything, levels of
GAL4DB-ASF/SF2 found in extracts testing mutants m1 to m5 were higher
than those for the
271-437 (data not shown). This experiment also
suggested that yeast cells have a kinase capable of phosphorylating the
RS domain of ASF/SF2, given that mAb104 exclusively recognizes
phosphorylated RS domains (data not shown).
Phosphorylation of the RS1nc Segment Correlates with ASF/SF2
Binding--
Arginine to lysine substitutions within the RS1nc segment
abolished binding to ASF/SF2 in the two-hybrid system (Figs.
3B and 4B) and in a far Western assay (see Fig.
6). We asked whether or not these substitutions could lead to change in
the phosphorylation of this segment of U1 70k. We cloned DNA fragments
encoding RS1nc or RS1nc mutants m1, m3, and m7 (Fig. 4A)
into the pGEX-2TK bacterial expression vector. Fusion proteins
GST-RS1nc, GST-RS1nc (M1), GST-RS1nc (M3), and GST-RS1nc (M7),
respectively, were overexpressed, purified, and phosphorylated as
described under "Materials and Methods." Whereas GST protein was
not phosphorylated by SRPK1, GST-RS1nc was efficiently phosphorylated
by this enzyme (lanes 1 and 2, Fig.
4C). We have also observed phosphorylation of full-length native U1 70k by recombinant SRPK1 (data not shown). The
phosphorylation of the mutant fusion proteins was significantly
diminished (lanes 3-5, Fig. 4C, top
panel). The same results were obtained if S100 HeLa cell extract
was used as the source of kinase activity (49). The fusion proteins
were present in comparable amounts in the glutathione-agarose beads
used in the kinase assays (Fig. 4C, bottom
panel). Interestingly, preliminary experiments have shown that the
SRPK1 phosphorylated GST-RS1nc can bind a native SRp30 protein
considerably better than the nonphosphorylated GST-RS1nc (data not
shown). These data establish a correlation between phosphorylation of
the RS1nc segment and efficient binding to ASF/SF2.
The RS1nc Segment of U1 70k Interacts with the RS Domain of
ASF/SF2--
Previously we and others demonstrated that the
interaction of the U1 70k protein with ASF/SF2 depended on the RS
domain of the latter (9, 38). Moreover, we have previously shown that the deletion of the C-terminal half of the RS domain in ASF/SF2 did not
eliminate this binding (9, 38). We used the two-hybrid system to
evaluate the binding of several regions of the U1 70k to ASF/SF2,
ASF
RS, which does not contain an RS domain, and to isolated ASF/SF2
RS domains. U1 70k bound to ASF/SF2, ASF-RS, and ASF-RSn equally well,
to ASF-RSc somewhat less, and to ASF
RS not at all (Table I). The U1
70k RS1n and RS1nc followed a very similar pattern, demonstrating a
requirement for the RS domain of ASF/SF2 in all these interactions
(Table I). As expected, RS2, RS1c and RS1nn did not show significant
binding to any of the ASF/SF2 constructs (Table I). It is likely,
therefore, that the binding of ASF/SF2 observed with the short segments
of U1 70k is mediated by the same determinants of specificity that
mediate binding of both full-length proteins. Given that the segment
spanning residues 248-270 binds ASF/SF2 efficiently and with
specificity and that it is the most arginine/serine-rich sequence in U1
70k, we suggest that this is the equivalent of the RS domain of the U1
70k protein.
The U1 70k Protein Can Interact with a Repeat of Five
Serine/Arginine Dipeptides--
A fusion protein in which the
GAL4-binding domain was connected to a repeat of five Ser-Arg pairs
(SR)5 was tested in the two-hybrid system for binding to U1
70k and several deletion mutants (Fig.
5). The (SR)5 fusion protein
bound the U1 70k, RS1n and RS1nc, but not RS1c and RS1nn, exactly as
seen for full-length ASF/SF2 and for the RS domain of ASF/SF2 (ASF-RS)
(Fig. 5 and data not shown). As expected, the (SR)5 fusion
protein was also shown to bind ASF/SF2 and ASF-RS, which were in this
case produced as GAL4AD fusion proteins (Fig. 5). Thus the
(SR)5 "synthetic domain" was capable of recapitulating
the binding of the authentic ASF/SF2 RS domain in the two-hybrid
system.

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Fig. 5.
A synthetic (SR)5 domain can
interact with the RS1nc segment of U1 70k. The relative strength
of interactions between U1 70k or variants and a synthetic domain of
five serine/arginine repeats (SR)5 was determined by
measuring -galactosidase activity. Quantitative liquid
-galactosidase assay were performed on at least three independent
experiments for each combination, and the mean values are shown in the
bar graph. The level of background -galactosidase activity obtained
with the negative control for the two-hybrid system was set at 1.0. The
values ± S.D. were: for U1 70k, 10.3 ± 5.6; for RS1n,
6.2 ± 1.9; for RS1c, 0.9 ± 0.0; for RS1nn, 1.0 ± 0.1;
for RS1nc, 5.7 ± 1.3.
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The RS1nc Segment Can Bind ASF/SF2 in a Far Western Assay--
In
prior work Khotz et al. (9), showed that the C-terminal half
of U1 70k was required for binding ASF/SF2 in a far Western assay. We
used a similar assay to confirm the results obtained with the
two-hybrid system. Yeast extracts were subjected to SDS-PAGE, and the
electrophoretically separated proteins were immobilized and probed with
radiolabeled phosphorylated ASF/SF2 (see "Materials and Methods").
ASF/SF2 bound U1 70k and RS1nc but not
RS1nc, RS1nc-mut, and mutants
m1 to m5 in this far Western assay (Fig. 6A). The same yeast extracts
were subjected to conventional Western assays with the anti-U1 70k
murine monoclonal antibody described in Ref. 46 to demonstrate that
levels of the fusion proteins were approximately the same. The levels
of
RS1nc and the mutants m1 to m5, all of which have the epitope
recognized by the monoclonal antibody were observed to be present at
levels even higher than the full-length 70k (Fig. 6B). The
smaller RS1nc and RS1nc-mut proteins could not be detected with this
antibody. The results of the far Western assay confirmed those obtained
with the two-hybrid system, indicating that the segment of U1 70k
spanning amino acids 240-270 is both necessary and sufficient for an
efficient interaction with ASF/SF2.

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Fig. 6.
Sequences within the RS1 region are necessary
and sufficient to bind ASF/SF2 in a far Western assay.
A, yeast extracts from cells overexpressing U1 70k, ASF/SF2,
or variants of these were subjected to far Western assays using
radiolabeled ASF/SF2 as a probe. 40 µg of total protein was loaded
from extracts overexpressing the following fusion proteins: murine
p53-Gal4BD (P, lane 1); SV40 Tag-Gal4AD
(N, lane 2); U1 70k (lane 3); RS1nc
(lane 4); RS1nc (lane 5); m1-m5 mutants
(lanes 6-10); RS1nc-mut (lane 11). B,
yeast extracts from cells overexpressing U1 70k, ASF/SF2, or variants
of these were subjected to Western assays using the monoclonal antibody
to the U1 70k protein. The signals were obtained using using a
secondary antibody to murine Ig linked to horseradish peroxidase in an
ECL assay.
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DISCUSSION |
The U1 70k protein binds ASF/SF2 via the RS domain in this protein
(9, 38) and in so doing promotes the incorporation of the U1 snRNP and
ASF/SF2 to a trimeric complex with pre-mRNA (8, 9). In this
manuscript we have mapped a region within the U1 70k protein that
mediates ASF/SF2 binding. The RS1nc segment, which encompasses amino
acids 248-270, contains two copies of the sequence "RRRSRSRD." We
propose that this segment is a minimal RS-like domain in the U1 70k
protein. Sequences at the C terminus of the RS1 region
(289RKRRSSRSRE297) probably play an ancillary
role in binding of ASF/SF2, and thus it may be that the RS1 region may
be the full RS domain.
The two RRRSRSRD repeats in close proximity, within RS1nc, represent
the most arginine/serine-rich region in U1 70k. Our mutational analysis
clearly shows that Arg269 was critical for efficient
ASF/SF2 binding. The fact that Arg269 was also essential
for phosphorylation of the RS1nc segment suggests that one of the roles
of the arginines could be to direct the phosphorylation of the
adjoining serines by kinases with RS specificity. Comparison of the
sequence of U1 70k homologues from human to yeast reveals remarkable
conservation of the RS1nc segment (Fig. 7); Arginines 256, 258, and 260 (human
numbering, see Fig. 1A) are conserved in human, toad
(Xenopus laevis), mouse, fly (D. melanogaster),
and Arabidopsis thaliana (Fig. 7). The latter two arginines
are also conserved in yeast (S. cerevisiae) (Fig. 7). In the
sequence of D. melanogaster there appears to have been a
duplication of this sequence. The N-terminal RSRSR pentapeptide in all
animal species in Fig. 7 is flanked at the C terminus by an acidic
amino acid. At the N terminus there is a propensity to find glutamic
acid followed by two arginines immediately preceding the N-terminal
pentapeptide (Fig. 7). The C-terminal pentapeptide is less highly
conserved; however, a tendency toward the same pattern described can
also be noted.

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Fig. 7.
Phylogenetic sequence comparison of the RS
domain of U1 70k protein. The sequence of the minimal RS-like
domain of the human U1 70k is shown (10). The sequences of U1 70k
homologues in Mus musculus (mouse) (91868), X. laevis (toad) (P09406), D. melanogaster (fly) (M31162),
A. thaliana (U52909), and S. cerevisiae (yeast)
(Q00916) were aligned using the program PILEUP (50), except for the
sequence denoted drosophila 70k1, which was aligned
by inspection. Underlined residues are those that appear in
at least six of the seven sequences.
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The recent discoveries of plant and fungal SR proteins and SRPK1-like
kinases fit well with the scenario of a conserved region in U1 70k
designed for interactions with SR proteins. This interaction must not
be essential given that the predicted RS-like domain homology lies
within the C-terminal 80 amino acids in the yeast U1 70k, and these are
dispensable for viability (15). This, however, does not preclude
conditions in which such an interaction may be important. It is
worthwhile noting that the RNA-binding domain of this protein was also
shown to be dispensable for growth (15). The relevance of the
interaction between SR proteins and the U1 70k needs to be studied
in vivo. Our experiments with human U1 70k and the
phylogenetic comparison suggest strong conservation of the RS-like
domain in animal species. Modest conservation of this region in
S. cerevisiae and A. thaliana suggests an
ancestral origin for this domain and the possibility that these
sequences may be employed for SR protein binding in these
organisms.
The most conserved feature of these sequences is the alternating
positioning of arginines, which may indicate a relationship with RD/RE
domains in human U1 70k and other proteins (14). The general solution
for protein-protein interaction encoded in these alternating arginines
may be similar in all of these domains. Yet it is clear from our data
that the RD/RE domains of U1 70k (e.g. RS1nn) cannot
substitute for the RS-like domain, and therefore the details of
recognition must be specific to the latter. Modeling RS-RS binding is
only speculative; however, certain possibilities are made less likely
given our data and that of others. Hydrogen bonding between
arginines and serines is unlikely given recent data that these
proteins bind better when the serines are phosphorylated (49). Ion
bridges between arginines and phosphorylated serines are also unlikely
given the dramatic inhibitory effect seen upon substituting lysine for
arginine. It is thus more plausible that the arginines may interact
with each other, which could be mediated by hydrogen bonding or
stacking. The phosphorylated serines in the RS repeats or the charged
acidic amino acids in the RE/RD repeats may provide important
structural contributions to this binding. Moreover, phosphorylation of
the serines, which enhances the binding between ASF/SF2 and U1 70k (49)
and is required for splicing (51), could provide for regulation of
these protein-protein interactions. Further mutational analysis
complemented by structural studies should put these ideas to
experimental test.
We thank Russ Carstens, Zvi Pasman, and Qing
Chen for helpful comments on this manuscript and members of the
Garcia-Blanco laboratory for insightful discussions. We also thank
Sabina W. Sager for assistance in the preparation of this
manuscript.