A Serine/Arginine-rich Domain in the Human U1 70k Protein Is Necessary and Sufficient for ASF/SF2 Binding*

Wenhong CaoDagger and Mariano A. Garcia-BlancoDagger §parallel

From the Departments of Dagger  Pharmacology and Cancer Biology, § Microbiology, and  Medicine, Levine Science Research Center, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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. ASFDelta 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. Delta 271-437, Delta 249-437, and Delta 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-Delta 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 Delta 271-437 (m1 to m5) were made by cloning annealed oligonucleotides so as to extend the open reading frame in construct Delta 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 beta -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 [gamma -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 [gamma -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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 beta -galactosidase activity. Quantitative liquid beta -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 beta -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, Delta 271-437, Delta 249-437, and Delta 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.

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 (Delta 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 (Delta 249-437), which eliminates two runs of RS dipeptides, completely abolished binding and, as expected, so did deletion to amino acid 230 (Delta 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 beta -galactosidase activity. Quantitative liquid beta -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 beta -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 beta -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.

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 (Delta 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 Delta 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 Delta 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 beta -galactosidase activity. Quantitative liquid beta -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 beta -galactosidase activity obtained with the negative control was set at 1.0.

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 (Delta 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 Delta 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 Delta 271-437 or variants and ASF/SF2 was determined by measuring beta -galactosidase activity. Quantitative liquid beta -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 beta -galactosidase activity obtained with the negative control was set at 1.0. The values ± S.D. were: for Delta 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).

We constructed a library of variants of the clone Delta 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 (Delta 271-437) and five mutants were obtained and analyzed. Three mutants (m2, m3, and m5) had double Arg right-arrow Lys substitutions that changed both RSRSR pentapeptides. All of these reduced ASF binding to ~25% of the Delta 271-437 (Fig. 4B). This level of binding was clearly above that obtained with Delta 249-437, a mutant missing the segment with the two pentapeptides (Fig. 1B), suggesting that the Arg right-arrow 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 Delta 271-437 (wild type) and the mutants m1 to m5 were approximately the same, as were the levels of Delta 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 Delta 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, ASFDelta 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 ASFDelta 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 beta -galactosidase activity. Quantitative liquid beta -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 beta -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.

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 Delta 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 Delta 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); Delta 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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This study was funded by a grant from the National Institutes of Health (to M. A. G-B.). The Levine Science Research Center is supported by the Keck Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Box 3686, DUMC, Durham, NC, 27710. Tel.: 919-613-8632; Fax: 919-613-8646; E-mail garci001{at}mc.duke.edu.

The abbreviations used are: snRNP, small nuclear protein; RS, arginine/serine; SR, serine/arginine; GST, glutathione S-transferasePAGE, polyacrylamide gel electrophoresis.
    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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