INTRODUCTION

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A complex array of general transcription factors, DNA binding activators and repressors, and a multitude of coregulators mediate transcription in eukaryotes (32, 48, 49). Regulation of the frequency of transcription initiation is a well-documented means of controlling gene expression (32, 48, 64), but many genes are also controlled by modulation of the ability of RNA polymerase II (RNAPII) to elongate transcripts (11, 15-17, 37, 53, 82, 93). Although the mechanisms controlling RNAPII elongation efficiency are not completely understood, it is clear that the interplay of multiple protein factors regulates RNAPII elongation efficiency (21, 70, 75). Some of these elongation factors have been identified, and they belong to two classes: positive transcription elongation factors (P-TEFs), such as positive elongation factor b (P-TEFb) (38, 47, 67, 68), and negative transcription elongation factors (N-TEFs), such as DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) (28, 84, 90). In addition to trans-acting elongation factors, nucleic acid sequences in the template and transcript can modulate elongation (11, 44, 82). A topic central to elongation control is the role of the carboxyl-terminal domain (CTD) of the largest subunit of RNAPII (25). The CTD contains 52 repeats of a 7-amino-acid sequence with the consensus YSPTSPS and is the substrate for several kinases, including P-TEFb (25, 68, 99). Phosphorylation of the CTD occurs during a transitional step of the transcription cycle, the switch from the initiation phase to the elongation phase. Hypophosphorylated RNAPII (designated RNAPIIA) is preferentially recruited to a promoter and initiates transcription. Subsequently, the RNAPII becomes hyperphosphorylated (RNAPIIO) as it clears the promoter. The CTD acts as a platform that recruits regulatory factors to RNAPII transcription complexes, and the phosphorylation of the CTD serves as a switch to regulate this recruitment. Regulatory initiation factors, such as the Mediator complex, bind to the hypophosphorylated CTD (48, 60). CTD phosphorylation causes the release of these initiation factors from the CTD and allows elongation factors to bind (65, 87). The phosphorylated CTD (phospho-CTD) can also recruit pre-mRNA processing proteins, which include factors involved in 5'-end cap formation (e.g., capping enzyme) and splicing (e.g., the U1 small nuclear ribonucleoprotein snRNP-associated protein Prp40) (10, 22, 27, 30, 34, 35, 39, 54, 56, 58, 59, 62, 76, 94). The CTD also functions in 3'-end formation events (34, 55). Thus, the CTD of RNAPII serves as a control center for regulating transcript initiation, elongation, and processing. There are compelling reports that these processes are functionally coupled in cells and extracts (4, 23, 24, 29, 96).

The human transcription factor CA150 is a negative regulator of RNAPII transcription elongation (80, 81). Overexpression of CA150 in human cells inhibits transcript elongation in a promoter-specific manner, affecting the human immunodeficiency virus long terminal repeat and the human ₄-integrin promoter while having no influence on several other viral promoters (such as simian virus 40 and cytomegalovirus promoters) (80). The specificity of the repression is dictated by core promoter elements, mainly the TATA box (80), which has also been observed for Tat-mediated activation of elongation (15, 50, 63). Although the mechanism of CA150-mediated repression is not known, an insight into this process was the discovery that CA150 binds directly to the phospho-CTD of RNAPII (18). The primary sequence of CA150 contains several types of domains that characteristically function to mediate protein-protein interactions. Within the carboxyl-terminal half of CA150, there are six repeats of a recently identified sequence element termed the FF repeat motif, so called because of flanking conserved phenylalanine residues (7, 18). The FF repeats are protein interaction modules, about 50 amino acids in length, which have a predicted -helical structure. It was shown that the CA150 FF repeats are responsible for binding to the phospho-CTD of RNAPII (18). This finding led us to hypothesize that CA150 may target RNAPII elongation complexes by binding to the phosphorylated CTD.

The amino-terminal half of CA150 contains three WW domains. These domains are versatile protein interaction modules, about 35 amino acids in length, that form a stable triple-stranded, antiparallel, -sheet structure (77-79, 95). These compact WW domains interact with specific types of short proline-rich polypeptide sequences. Four classes of proline-rich ligands have been identified for WW domains: PPXY, PPLP, PR, and phospho-SP/phospho-TP (9, 78). Individual WW domains generally recognize one type of ligand, and the determinants of WW domain specificity are only now becoming understood (51, 83, 95). The ligands of the CA150 WW domains and their function in transcription were not known before the findings presented here.

In this study, we assessed the function of the protein interaction domains of CA150 in transcription. First, we tested whether the CA150 FF repeats, which bind to the phosphorylated CTD of RNAPII, were required for CA150-mediated repression of transcription. Deletion of the FF repeats caused a loss of function in repression, suggesting that interaction with the phospho-CTD was a key component in the pathway of CA150-mediated repression. Additionally, the amino-terminal half of CA150, which contains the WW domains, was necessary for efficient repression. We determined that WW1 and WW2 were important for CA150-mediated repression while WW3 was not required. This result led us to hypothesize that the CA150 WW1 and WW2 domains may bind to factors necessary for repression. To identify this protein(s), we used a binding assay to purify a protein from HeLa cells that interacted specifically with the CA150 WW1 and WW2 domains. This protein, splicing factor 1 (SF1), was originally identified as a constitutive splicing factor (40, 43); however, it has also been shown to repress transcription (97, 98). We have mapped the CA150 binding site in SF1 to a small proline-rich motif present in the carboxyl-terminus. This sequence may represent a new class of WW ligand. Finally, we compared the ligand binding specificity of CA150 WW domains to that of other classes of WW domains and ligands. The implications of these findings in relation to transcription regulation and pre-mRNA splicing are discussed.

	MATERIALS AND METHODS

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Plasmids. All glutathione S-transferase (GST) fusion constructs were made in the pGEX2TK vector (Amersham-Phamacia) by cloning PCR products into the BamHI and EcoRI sites. PCR cloning was done with Pfu-Turbo (Stratagene) or Bio-X-Act (Denville Scientific) DNA polymerases. The pGEX2TK-N-CA150 construct contains amino acids 235 to 631 of CA150 fused to GST. pGEX2TK-WW1 contains CA150 amino acids 129 to 169, pGEX2TK-WW2 contains amino acids 427 to 467, and pGEX2TK-WW3 contains amino acids 526 to 566. CA150 expression constructs were cloned by inserting CA150 PCR products with BglII ends into the BamHI site of the mammalian expression vector pEFBOST7, which contains an amino-terminal T7 epitope tag. pEFBOST7 CA150 was described previously (80). SF1 constructs were subcloned from the SF1-Bo isoform, provided by Angela Kramer (Université de Genève, Geneva, Switzerland), by inserting SF1 PCR products into the BamHI and XbaI sites of the pcDNA3.1HisC mammalian expression vector (Invitrogen). This vector contains both His₆ and T7 epitope tags at the amino terminus. The SF1-Bo isoform is identical to HeLa SF1 (SF1-HL1 and HL2) except for 42 amino acids at the carboxyl terminus (2, 42). LacZ-SF1(aa420-500) containing amino acids 420 to 500 of SF1-Bo and LacZ-SF1(aa461-500) containing amino acids 461 to 500 of SF1 were cloned by insertion of SF1 PCR fragments with KpnI ends into pcDNA3.1HisBLacZ (Invitrogen). The ₄-integrin promoter reporter gene p(300)Alpha-4CAT and the transfection efficiency control reporter pTK-LUC were described previously (80). The pcDNA3.1HisC-LacZ transfection efficiency control plasmid was purchased from Invitrogen.

Site-directed mutagenesis. Site-directed mutagenesis of CA150 WW domains was performed using the Quickchange (Stratagene) method as specified by the manufacturer. For the mutation of the WW domains in CA150, the following constructs were generated by site-directed mutagenesis: pEFBOST7-CA150 WW1mt had amino acids Y148 Y149 Y150 changed to AAA, pEFBOST7-CA150 WW2mt had amino acids Y446 Y447 Y448 changed to AAA, pEFBOST7-CA150 WW3 had amino acids F545 F546 Y547 changed to AAA, and pEFBOST7-CA150 WW1mt+WW2mt had amino acids Y148 Y149 Y150 changed to AAA and amino acids Y446 Y447 Y448 changed to AAA. The GST expression vector pGEXTK-N-CA150 was used as a template to create WW2 or WW3 mutants in the N-CA150 far-Western probe. pGEX2TK-N-CA150 WW2mt was created by mutating amino acids Y446 Y447 Y448 to AAA. pGEX2TK-N-CA150 WW3mt was created by changing amino acids F545 F546 Y547 to AAA.

GST fusion protein purification. GST fusion proteins were purified from Escherichia coli BL21 (Stratagene) grown to an optical density at 600 nm of 0.6 and then induced for 2 h using 0.1 mM isopropyl--D-thiogalactopyranoside (IPTG). Cells were lysed with lysozyme in 1× phosphate-buffered saline (PBS) and sonication three times for 30 s at 10 W. Triton X-100 was added to a final concentration of 1%. Cell debris was removed by centrifugation at 10,000 × g for 30 min. A 1-ml bed volume of glutathione-agarose beads (Sigma) was added to the supernatant and allowed to bind for 1 h at 4°C. The beads were washed three times with 1× PBS containing 1 M NaCl. Purified proteins were eluted from the beads in 50 mM Tris-HCl (pH 8.0) containing 10 mM glutathione. Protein concentrations were quantified by the Bradford assay (Bio-Rad).

Immunoprecipitations. GST and CA150 antibodies were antigen affinity purified from the same rabbit polyclonal serum (raised against a GST-CA150 fusion protein) by the method of Harlow and Lane (33). A 50-µl volume of HeLa cell nuclear extract was diluted to 200 µl (final volume) with IP buffer (20 mM HEPES [pH 7.9], 150 mM KCl, 20% glycerol, 1 mM dithiothreitol, 1% Triton- X-100, 0.5% NP-40, 0.2 mM EDTA). Then 15 µg of each antibody was added to the diluted nuclear extract, and the mixture was incubated with end-over-end rotation at 4°C for 4 h. Immune complexes were collected with 500 to 750 µg of magnetic protein A beads (BioMag protein A beads; Perceptive Biosciences) and a magnet. The pellets were washed four times with 1 ml of IP buffer by rotating for 5 min at 4°C. The pellets were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (12.5% polyacrylamide) and analyzed by Western blotting with anti-CA150 antibody and by far-Western blotting with the N-CA150 probe.

Western and far-Western blotting. To prepare the membranes for use in the Western and far-Western assays, protein samples were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Western detection was performed using standard techniques. The monoclonal anti-T7 antibody (Novagen) or anti-HisG (Invitrogen) was used to detect T7 or His₆ epitope-tagged proteins, respectively. CA150 was detected using rabbit polyclonal antibodies. Enhanced chemiluminescence (Amersham-Pharmacia) was used for all Western blot detections.

Transfection of 293T cells. The human cell line 293T was used in all cell culture experiments. Transfections were performed using Lipofectamine as specified by the manufacturer (Gibco BRL). Cells were grown on BioCoat poly-D-lysine six-well plates (Beckton-Dickinson) for transfection. Liposomes were formed and added to the cells for 5 h. For functional assays, 1.5 µg of the p(300)Alpha-4CAT was used as a reporter construct along with either 10 ng of pHSV-TK-Luc or pcDNA-LacZ as transfection efficiency controls. The amounts of CA150 expression constructs were carefully controlled by using equimolar amounts of the constructs in each transfection, with the remaining mass of DNA transfected always balanced with carrier tRNA. Portions (2 µg) of pEFBOST7-CA150 expression plasmids or pEFBOST7 vector control were cotransfected with the reporter plasmids. Following transfection, the cells were grown for 48 h and then harvested for enzyme assays. The cells were washed twice in PBS and then resuspended in 150 µl of 50 mM Tris-HCl(pH 8.0). WCL were prepared by three cycles of freezing and thawing rapidly from 80 to 37°C. Cellular debris was removed by centrifuging at 20,000 × g for 5 min at 4°C. The supernatant was then removed and stored as the WCL.

Reporter gene assays. Chloramphenicol acetyltransferase (CAT) and luciferase assays were performed as described previously (80). -Galactosidase activity was measured using chlorophenol rad--D-galactopyranoside (CPRG) substrate. A 10-µg portion of WCL was mixed with CPRG reagent and incubated for 7 min at 25°C. Reactions were stopped by addition of 0.5 M Na₂CO₃. The product was measured at a wavelength of 574 nm. CAT reporter gene activity was measured in at least three independent samples for each CA150 test construct. CAT activities were measured and corrected for transfection efficiency by dividing the CAT activity by the luciferase or -galactosidase activity. The percent inhibition was determined by first calculating the fold inhibition of corrected CAT activity of wild-type CA150 in relation to the vector control, which was on average 5-to 10-fold inhibition. The fold inhibition by wild-type CA150 was set as 100% inhibition. The percent inhibition of the CA150 mutation and deletion test constructs was then calculated relative to that of the wild-type CA150.

Chromatography and microsequencing. All chromatography was performed using a Pharmacia fast protein liquid chromatography system. Protease inhibitor Complete tablets (Boehringer-Mannheim) were used in all buffers. Fractions were assayed for CIP80 by far-Western analysis with the N-CA150 probe. A phosphocellulose P11 (Whatmann) column with a 50-ml bed volume was equilibrated in HEK100 (HEPES [pH 7.9], 10 mM EDTA, 100 mM KCl). Then 30 ml of HeLa nuclear extract was applied to the column, and bound proteins were eluted by sequential washes with the following gradient: HEK250, HEK500, HEK750, and HEK1000. The 500 mM KCl fraction contained the CIP80 peak. The fraction was dialyzed into HEK100 and applied to a 5-ml-bed-volume HighS column (Bio-Rad). Proteins were eluted with a linear gradient from 100 to 1,000 mM KCl, Coomassie stained, and assayed by far-Western blotting with the N-CA150 probe. A 300-µl volume of the CIP80 peak fraction was precipitated using 2 volumes of acetone and then separated by SDS-PAGE. The 80-kDa band corresponding to CIP80, visualized by Coomassie staining, was excised from the gel and used for microsequencing. Protein microsequencing analysis was performed by John Leszyk (Protein Microsequencing Laboratory, University of Massachusetts Medical School, Shrewsbury, Mass.). A tryptic digest of CIP80 was analyzed by matrix-assisted laser desorption mass spectrometry (MALDI-MS). Nine peptides were identified and sequenced, all of which belong to the protein SF1 (and also designated ZFM1, mBBP, and ZFP162) (2, 41, 42, 88). Peptide sequences were confirmed by mass spectrometry and post-source decay fragmentation analysis.

	RESULTS

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The phospho-CTD binding sites of CA150 are required for repression of transcription. We performed a structure-function analysis of several of the protein interaction domains of CA150 to determine their role in CA150-mediated repression. The functional assay that we used involves a CAT reporter gene, whose expression is controlled by the human ₄-integrin promoter (80). The ₄-integrin promoter is controlled by both DNA binding activators and repressors and is regulated during hematopoeitic development and differentiation (3, 26, 45, 46, 72). Previously we demonstrated that CA150 inhibits the ₄-integrin promoter in a dose-dependent manner and that core promoter elements, containing the TATA box, were required for mediating this effect. We had also previously shown that the FF repeats of CA150 mediate binding to the phosphorylated CTD of RNAPII (18). To assess the function of the FF repeats, a CA150 construct with a deletion of all six repeats, CA150(1-663), was created and tested for repression of ₄-integrin (Fig. 1). CA150(1-663) exhibited a 70% loss in repression activity compared to full-length CA150. Thus, the FF repeats are necessary for efficient CA150-mediated repression. These results are consistent with the view that the interaction of CA150 with the phospho-CTD of elongating RNAPII is required for efficient repression. It was not clear, however, whether CTD binding is sufficient for this activity. For instance, the repression by CA150 could conceivably be the result of competitive binding between CA150 FF repeats and other elongation factors for binding to the phospho-CTD. To test this possibility, a construct, CA150(590-1098), that contains all six FF repeats but is lacking the N-terminal half of the protein was assayed for repression. CA150(590-1098) had greatly diminished activity relative to full-length CA150, exhibiting an 80% loss of repression (Fig. 1). This result demonstrates that the FF repeats are not sufficient for repression. The expression of the variant CA150 proteins was confirmed by Western blot analysis (Fig. 1). These CA150 proteins all contain the nuclear localization signal and were shown to be localized in the nucleoplasm, as assayed by immunofluorescence using an antibody against the amino-terminal T7 epitope tag of each CA150 construct (data not shown). We have also determined that the FF repeats were necessary for direct binding to phospho-CTD; full-length CA150 bound to phospho-CTD, while CA150(1-663) did not (A. C. Goldstrohm, S. Carty, A. Greenleaf, and M. Garcia-Blanco, unpublished data). Moreover, CA150(590-1098) was shown to coimmunoprecipitate RNAPII as well as full-length CA150 did (18). Therefore, the FF repeats appear to be necessary and sufficient for phospho-CTD binding whereas they are necessary but not sufficient for efficient repression of transcription.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   The carboxyl-terminal and amino-terminal halves of CA150, containing FF repeats and WW domains, respectively, are required for repression of the 4-integrin promoter. CA150 test constructs were overexpressed in 293T cells with an 4-integrin promoter driving the expression of the CAT reporter gene to assay CA150-mediated repression. CA150 constructs shown on the left indicate the domains contained in each protein. CA150 WT is the wild-type 1,098-amino-acid protein. CA150(1-663) and CA150(590-1098) contain amino acids 1 to 663 and 590 to 1098, respectively. CA150 protein domain abbreviations: PP, polyproline-rich region; WW, WW domain; QA, glutamine-alanine repeats; NLS, nuclear localization signal; FF, FF repeats. The percent inhibition of CAT activity was calculated as described in Materials and Methods. The activity of each construct was assayed at least three times independently, and the error bars represent the standard deviation. The right panel is a representative Western blot, against the amino-terminal T7 epitope tag of each protein, showing the expression of each construct.

The WW domains of CA150 are required for efficient repression. The loss of repression by CA150(590-1098) (Fig. 1) demonstrated that important determinants of CA150 function exist in the amino-terminal half of the protein. We hypothesized that CA150 may inhibit transcription by binding to the RNAPII CTD via the FF repeats and recruiting a repressor(s) to the transcription complex through the protein interaction domains in its amino-terminal half. The three amino-terminal WW domains of CA150 were strong candidates for such a function (CA150 WW domains are shown in Fig. 2A). We created point mutations in or deletions of each WW domain of CA150 and tested these constructs for repression activity. Since the results for mutations and deletions are nearly identical, we present only the data for the point mutations of CA150 WW domains in Fig. 2B. The highly conserved central aromatic amino acids of each WW domain were mutated to alanine (WW1 and WW2, YYY to AAA; WW3, FFY to AAA [Fig. 2]). These three aromatic amino acids are part of the proline binding pocket of WW domains (36, 83, 95). It has been shown that mutation of these residues does not destabilize the domain; therefore it is likely that the mutated WW domains were folded correctly (51). The mutated CA150 constructs were then tested for repression of the ₄-integrin promoter. Mutation of WW1 (CA150 WW1mt) and WW2 (CA150 WW2mt) reproducibly resulted in 35 and 38% loss of activity, respectively, while mutation of WW3 had no effect on repression (Fig. 2B). Although the effect of disrupting WW1 and WW2 was modest, the results were highly reproducible. These data suggest that WW1 and WW2 contribute to CA150-mediated repression. We also created a double mutation of both WW1 and WW2 (CA150 WW1mt+WW2mt), which caused a 63% loss of activity relative to wild type, further supporting the requirement of WW1 and WW2 for repression (Fig. 2B). To evaluate a possible ancillary role for WW3, we disrupted WW1 and WW3 in combination and WW2 and WW3 in combination. These combinations behaved as the single WW1 and WW2 disruptions, suggesting that WW3 did not play a supportive role in the repression activity. CA150 protein expression levels from each construct were essentially identical as determined by Western blotting against the amino-terminal T7 epitope tag of each construct (Fig. 2B). In addition, we found that replacement of the CA150 WW1 domain with a functional WW domain from another protein (YAP) (52, 89), which has a ligand specificity distinct from that of CA150 WW1 and WW2 (for instance, see Fig. 5), also resulted in a loss of CA150 activity, identical to deletion or mutation of CA150 WW1 domain (data not shown). Therefore, the WW1 and WW2 domains of CA150 are specifically required for the repression activity. These results indicate that factors that interact with WW1 and WW2 are important for CA150-mediated repression.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   The WW1 and WW2 domains of CA150 are required for efficient repression of the 4-integrin promoter. (A) Sequence comparison of CA150 WW domains WW1, WW2, and WW3. Alignments were constructed using the Clustal alignment function of the Mac Vector 6.5.3 program. A bracket highlights the central aromatic residues of the domains, YYY and FFY, which constitute part of the proline recognition site. These are the amino acids that were mutated in the CA150 WW domain mutant constructs in panel B and Fig. 3A. (B) Mutation of WW1 or WW2 reduced CA150-mediated repression of the 4-integrin promoter relative to wild type. A double-mutant CA150, containing mutations in both WW1 and WW2 (CA150 WW1mt+WW2mt), resulted in an even greater loss of repression activity. The percent inhibition of CAT activity is represented in the graph at the right. The standard error is also indicated. The site-directed mutagenesis of each domain is described in Materials and Methods. Diagrams of each CA150 test construct are depicted. Abbreviations are the same as those used in Fig. 1. YYY-AAA indicates the mutations of tyrosine to alanine in the central aromatic residues of WW1 and WW2. FFY-AAA indicates mutations of phenylalanine and tyrosine to alanine in the central aromatic residues of WW3. The bottom panel shows a representative anti-T7 Western blot demonstrating equal levels of expression for each CA150 protein.

A protein of 80 kDa interacts with CA150 WW domains. Given the important role played by WW1 and WW2 in CA150-mediated repression, we sought to identify factors that interact with these domains. We chose to use a far-Western (protein interaction) blotting approach to identify CA150-interacting proteins (CIPs). WW domains are particularly amenable to this approach because they can autonomously fold into their native structure and their ligands are small proline-rich motifs that can easily renature. This type of analysis has been extensively used to characterize other WW domains and their ligands (9, 20). We created a GST fusion protein, designated N-CA150, containing the WW2 and WW3 domains, to be used as a probe for detecting CIPs (Fig. 3A). HeLa cell nuclear extract (NE) was separated by SDS-PAGE, blotted to a membrane and renatured, and probed with nanomolar concentrations of the N-CA150 or negative-control GST probes (Fig. 3B). As expected, GST did not interact with any proteins in NE. N-CA150 detected a strong interaction with a CIP of 80 kDa, tentatively named CIP80 (Fig. 3B). Several weaker CIPs were also observed, some of which varied among extract preparations. We chose to analyze CIP80 because it was consistently the strongest interaction observed, both by far-Western assay and coimmunoprecipitation analysis (see below).

View larger version (28K): [in this window] [in a new window]   FIG. 3.   A CA150-interacting protein of 80 kDa (CIP80), which interacts directly with CA150, is the splicing-transcription factor SF1. (A) Diagram showing the recombinant CA150 proteins used as probes in the far-Western analysis. Abbreviations are the same as those used in Fig. 1. N-CA150, WW2mt, and WW3mt contain amino acids 235 to 631 of CA150, except that the WW2mt probe had the central aromatic residues of WW2 domain mutated from YYY to AAA and WW3mt had the corresponding residues mutated from FFY to AAA. These mutations ablate the recognition of proline-containing ligands by the WW domains. (B) Far-Western analysis of HeLa nuclear extract with the N-CA150 probe, as indicated at the top of the figure, detected a CA150-interacting protein with an apparent mass of 80 kDa, designated CIP80. The negative-control far-Western blot with a GST probe did not bind to nuclear proteins, as expected. (C) Far-Western analysis of HeLa nuclear extract showed that mutation of the WW2 domain ablates binding to CIP80 while mutation of the WW3 domain has no effect. CA150 probes are indicated at the top, and CIP80 is indicated on the left. (D) Coimmunoprecipitation of CIP80 with CA150 from HeLa nuclear extract. CA150 was immunoprecipitated using antigen affinity-purified rabbit polyclonal antibodies, as indicated at the top. The immunoprecipitates were analyzed by Western blotting with anti-CA150 antibodies (top panel) and by far-Western blotting using the N-CA150 probe (bottom panel), as labeled on the left. (E) Purification scheme for isolating CIP80. The CIP80 protein was excised from an SDS-PAGE gel and microsequenced by mass spectrometry. CIP80 corresponds to the previously identified SF1 protein. (F) Fractions from HighS chromatography, developed with a linear gradient of 100 to 500 mM KCl, were analyzed by far-Western blotting with the N-CA150 probe. Molecular weight markers are indicated on the right of the figure in thousands. CIP80 is labeled on the left. The peak CIP80-containing fraction from the phosphocellulose step was included in the analysis and labeled Input. A 110-kDa protein that also interacts with the N-CA150 probe, albeit weakly, copurified with CIP80.

CIP80 is the splicing and transcription factor SF1. To determine the identity of CIP80, we purified it and obtained microsequence data. The purification protocol is described below and is outlined in Fig. 3E. HeLa nuclear extract containing CIP80 was separated by phosphocellulose chromatography, and fractions were assayed for the presence of CIP80 by far-Western blotting. CIP80 peak fractions were pooled and applied to a HighS column (Fig. 3F). The HighS peak fraction of CIP80 was then separated by SDS-PAGE, the 80-kDa band corresponding to CIP80 was excised from the gel, and peptides were generated and microsequenced using mass spectrometry. This analysis revealed that CIP80 is the previously identified pre-mRNA splicing-transcription factor SF1 (2, 12, 13, 40, 42, 69). Multiple isoforms of SF1 have been identified (see Discussion). The major isoform of SF1 in HeLa cells contains 638 amino acids and has a mobility of approximately 80 kDa on SDS-PAGE (2, 42, 69). In addition to its role in pre-mRNA splicing, SF1 is a transcription repressor, thus providing a possible explanation for its interaction with CA150 (97, 98).

View larger version (43K): [in this window] [in a new window]   FIG. 4.   Full-length SF1(1-638) was used to create a deletion series of the carboxyl-terminal half of the protein. The SF1 amino acids contained in each construct are indicated in parentheses. KH is the hnRNP K homology domain, and Zn represents the Zn knuckle motif. PP indicates the proline-rich region. (B) The CA150 WW1, WW2, and WW3 domain probes, represented in this diagram, were used in far-Western analysis. (C) Far-Western analysis of WCL from 293T cells overexpressing the SF1 constructs indicated at the top. HeLa NE was included as a positive control, and endogenous HeLa SF1 is indicated by an arrow on the left. 293T WCL transfected with empty vector or the lacZ expression vector were included as negative controls. Far-Western blotting of blots of these WCLs with each WW domain from CA150, as labeled on the left, detected protein-protein interactions with the overexpressed SF1 constructs. The bottom panel, labeled Western, is an anti-HisG Western blot of the overexpressed proteins that contained an amino-terminal His tag. Protein molecular weight markers are labeled on the right in thousands.

A proline-rich sequence in SF1 is sufficient for binding to CA150 WW domains. To characterize the ligand binding specificity of CA150 WW domains, we used the far-Western assay with recombinant WW1, WW2, and WW3 proteins and tested their ability to interact with prototypic peptides of the known classes of ligands (Fig. 5) (8, 9, 78, 79). As controls, we included WW domains with documented ligand specificity. Equal amounts of each purified GST-WW domain were separated by SDS-PAGE, transferred to membranes, and probed with the peptide ligands (Fig. 5). The WW2 domain of CA150 bound weakly to the PR motif-containing ligands; however, this binding was considerably weaker than that of FBP21 or FBP30, which exhibit high-affinity, specific binding to proline-arginine motifs (PR) (Fig. 5) (9). Thus, the PR motif is a poor ligand for WW2. The CA150 WW1 and WW3 domains did not bind any of these peptide ligands. In addition, some WW domains, including that of the Ess1 protein, can bind to the phospho-CTD of RNAPII (YpSPTpSPS ligand, where "p" indicates phosporylation of serine). The three WW domains of CA150 were tested for binding to phospho-CTD, and they did not bind (Goldstrohm et al., unpublished). These results indicate that the CA150 WW domains display a ligand binding specificity distinct from those of previously classified WW domains.

View larger version (63K): [in this window] [in a new window]   FIG. 5.   The ligand binding specificity of recombinant CA150 WW domains WW1, WW2, and WW3 was compared to that of WW domains with previously determined specificity. The identity of each WW domain is indicated at the top of the figure. Ligand binding specificities of the WW domains are as follows. The WW domain from the FBP11 protein specifically recognizes the PPLP motif from the ld10 protein. The FBP21 and FBP30 WW domains have a distinct binding preference for proline-arginine motifs (PR), such as those found in the SmB snRNP protein (designated SmB) and Sam68 (designated P3). The Pin1 WW domain binds specifically to motifs containing phosphoserine or phosphothreonine followed by a proline residue (phospho-SP/phospho-TP), such as that found in CDC25. Finally, the YAP WW domain interacts exclusively with a proline-rich sequence followed by tyrosine (PPPPY), which is found in WBP1. The top panel is a Coomassie-stained SDS-PAGE gel containing equal amounts of each WW domain. Purified GST protein served as a negative control. Peptides corresponding to the known classes of proline-rich ligand were used as probes in far-Western assays, shown in the bottom five panels. These probes are described in Materials and Methods. The identity of the proline-rich probes is indicated on the left, with the class of proline-rich motif indicated in parentheses. This analysis shows that CA150 WW domains did not bind strongly with the known classes of proline-rich ligands, indicating that they may recognize a novel proline-rich motif.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Amino acids 420 to 500 of SF1 are sufficient for binding to the WW2 domain of CA150. (A) The SF1 amino acids 461 to 500 or 420 to 500 were fused to the lacZ gene and expressed in 293T cells. WCL were prepared from these cells or control lacZ-transfected cells and subjected to far-Western analysis with the a WW2 domain probe, shown in the top panel. The expression constructs are indicated at the top of the figure. Anti-T7 Western blot analysis of the WCL demonstrates equivalent expression of each protein. (B) The CA150 binding site in SF1, composed of amino acids 420 to 500. The PPPxxQ motifs, which are the probable ligands of the WW1 and WW2 domains of CA150, are underlined.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

CA150 is a transcription repressor that inhibits the elongation efficiency of RNAPII in vivo (80) and in vitro (A. C. Goldstrohm and M. Garcia-Blanco, unpublished data). Previous work demonstrated that CA150 FF repeats interact directly and specifically with the phospho-CTD of RNAPII (18), and here we show that these repeats of CA150 are required for maximal repression. Taken together, these observations suggest that CA150 is targeted to the elongating RNAPII via the FF repeats-phospho CTD interaction. Once targeted to elongating RNAPII, CA150 can repress transcript elongation by one of several mechanisms. CA150 could potentially displace P-TEFs from the phospho-CTD; however, the fact that the FF repeats of CA150 are sufficient for phospho-CTD binding but not for repression in vivo (this study) or in vitro (Goldstrohm and Garcia-Blanco, unpublished) makes this possibility unlikely. CA150 could modify the enzymatic activity of RNAPII, as has been shown for the transcript cleavage-inducing factor TFIIS, which stimulates elongation by facilitating the release of the polymerase from an arrested state (86). Alternatively, CA150 could modify the activity of P-TEFs that also interact with RNAPII. We have observed that CA150 associates with multiple positive elongation factors including P-TEFb, Tat-SF1, and TFIIF (Goldstrohm and Garcia-Blanco, unpublished). CA150 may inhibit transcription by suppressing the action of these positive factors, or, conversely, these factors may associate with and counteract CA150. Finally, CA150 could mediate the recruitment of other effectors (N-TEFs) to the elongation complex. This possibility led us to look for other proteins that interacted with CA150.

The amino-terminal half of CA150 contains three WW domains, two of which, WW1 and WW2, are important for repression of transcription. We posited that these two domains could be important in recruiting other proteins that could collaborate with CA150. We identified a factor, SF1, which physically interacts with these WW domains. We found that the requirement of WW1 and WW2 domains for CA150-mediated repression correlated precisely with the binding specificity of WW1 and WW2 for SF1. These observations lead to two non-mutually exclusive suggestions about the role of SF1 on CA150 repression: CA150 recruits SF1 to act as an N-TEF, and/or CA150 uses SF1 to target the elongating RNAPII. It must be noted that we have not formally proven that the interaction with SF1 is required for CA150 function and thus we must also consider that the observed interaction is a surrogate interaction.

Could SF1 be an N-TEF? SF1 has indeed been shown to repress activation of transcription by certain chimeric transcription activators (97, 98). SF1 also represses transcription from a promoter when tethered to a Gal4 DNA binding domain and targeted to a promoter containing Gal4 binding sites (97). Taken together, these results suggest that recruitment of SF1 to a transcription complex, whether by a DNA binding factor or by CA150, can result in repression. The CA150 binding site resides in the proline-rich carboxyl terminus of SF1. Currently, the function of the proline-rich region is not known, and it has been reported to be dispensable for SF1 splicing activity (31, 69). Zhang and Childs found that a portion of this region of SF1 could bind to the activation domain of certain transcription factors, suggesting that the proline-rich region may function in transcription (97). At least 10 alternatively spliced SF1 isoforms have been identified, which vary in the proline-rich region and carboxyl terminus (2, 42; A. Kramer, personal communication). Several isoforms lack portions of the CA150 binding site (SF1 amino acids 420 to 500). So far we have determined that at least one of these isoforms, ZFM1-E12/E13 (42), interacts very weakly with CA150 in the far-Western assay, demonstrating that the CA150-SF1 interaction is isoform specific (data not shown). The ZFM1-E12/E13 isoform diverges from the major HeLa isoforms, SF1-HL1, SF1-HL2, and SF1-Bo, at amino acid 469, which resides within the CA150 binding site (42). Also, based on our binding analysis we predict that the SF1 isoform ZFM1-B3 would not bind to CA150, since it possesses an entirely different C -terminus beginning at residue 448 (42). In conclusion, the isoform-specific CA150-SF1 interaction advocates a possible mechanism for regulating CA150 activity depending on the repertoire of SF1 isoforms present in a cell. As an additional consideration, we note that two other mammalian proteins that possess WW domains have been shown to interact with SF1. The FBP11 protein, an ortholog of the U1-associated snRNP protein Prp40, and the U2 snRNP-associated protein FBP21 both bind to SF1. The binding site in SF1 for these proteins has not been mapped. However, because the WW domains from FBP11 and FBP21 exhibit different ligand specificities in comparison to CA150 (see Fig. 5), we expect that they do not compete with CA150 for binding to SF1.

Could SF1 target CA150 to elongating transcripts? SF1 binds RNA via its KH and Zn knuckle domains with rather poor sequence- specificity (2, 13). Could this RNA binding activity be a clue to its role in CA150-mediated repression? We propose that CA150 and SF1 together may target elongating RNAPII complexes by recognition of the two distinguishing characteristics of an RNAPII elongation complex: the phosphorylated CTD and the nascent RNA transcript. This idea is consistent with the pathway used by other elongation factors, which function by binding to the transcribing polymerase and the transcript. One example is the yeast Nrd1-Nab3 complex, which binds to the phospho-CTD of RNAPII (via Nrd1) and recognizes a sequence in the nascent RNA (both Nrd1 and Nab3 bind to RNA), leading to termination of transcription (22). One noteworthy difference between Nrd1 and CA150 repression mechanisms is that Nrd1 requires a specific cis-acting RNA sequence, designated U6R*, whereas we have not detected a transcript sequence specificity for CA150 activity. Another negative regulator of RNAPII elongation that may follow a similar pathway to that of CA150 is the multisubunit DSIF-NELF complexes. DSIF-NELF is responsible for inhibition of early elongation events, and this effect is reversed by P-TEFb (28, 85, 90). DSIF-NELF associates with RNAPII, mediated by the Spt5 subunit of DSIF, which binds directly to the large subunit of RNAPII (91). However, unlike CA150, DSIF-NELF binds to and inhibits unphosphorylated RNAPII (85, 90). The RD subunit of NELF has similarity to Nrd1 in that it contains a single RNA recognition motif that very probably binds to RNA, thus making it probable that DSIF-NELF contacts RNAPII and the nascent transcript to inhibit elongation (28, 90). Finally, prokaryotic RNAP is also regulated by elongation factors that bind the polymerase and the transcript. The transcription termination factor Rho interacts with the NusG protein to mediate termination. Rho also has an RNA binding domain that recognizes the nascent transcript, while NusG binds directly to the polymerase (71). In addition, the phage lambda N protein, an antitermination factor, affects elongation through a similar pathway. N is a positive elongation factor that binds to a cis-acting element in the nascent transcript, nut, and mediates antitermination in conjunction with cellular factors, including the NusA protein that binds directly to the elongating RNAP (57). Therefore, dual interactions with the polymerase and the nascent transcript may be a common mechanism used by elongation factors to identify their targets and carry out their function.

Does the CA150-SF1 interaction play a role in pre-mRNA splicing? Multiple studies have also demonstrated a role for SF1 in pre-mRNA splicing (1, 12, 13, 31, 40, 41, 69, 73, 74). The apparent dichotomy of function of SF1 in splicing and transcription can be viewed in two ways. SF1 may play independent roles in transcription and splicing, or it may participate in coupling the two processes (see below). The transcription assays used in our studies did not include introns; therefore, the transcription repression that we observe is not likely to be a splicing-related process. However, accumulating evidence suggests a role for CA150 in pre-mRNA splicing. Neubauer et al. discovered that purified spliceosomes contain CA150 (61). Likewise, we have observed that the snRNP Sm proteins coimmunoprecipitate with CA150, suggesting that CA150 can associate with snRNPs (Goldstrohm and Garcia-Blanco, unpublished). CA150 also coimmunoprecipitates with Tat-SF1 (Goldstrohm and Garcia-Blanco, unpublished), which, in addition to its ability to affect elongation, has been shown to be the mammalian ortholog of the splicing factor CUS2 and to associate with the splicing factor SF3a (66, 92). An interesting possibility is that the CA150-SF1 complex could function to coordinate the rates of transcript elongation and pre-mRNA processing. This idea is highly speculative, and future work is required to test it.