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. 2001 Nov;21(22):7617-28.
doi: 10.1128/MCB.21.22.7617-7628.2001.

The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1

A C Goldstrohm  1 , T R Albrecht, C Suñé, M T Bedford, M A Garcia-Blanco
Affiliations

The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1

A C Goldstrohm et al. Mol Cell Biol. 2001 Nov.

Abstract

CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the alpha(4)-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.

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Figures

FIG. 1
FIG. 1
The carboxyl-terminal and amino-terminal halves of CA150, containing FF repeats and WW domains, respectively, are required for repression of the α4-integrin promoter. CA150 test constructs were overexpressed in 293T cells with an α4-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.
FIG. 2
FIG. 2
The WW1 and WW2 domains of CA150 are required for efficient repression of the α4-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 α4-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.
FIG. 3
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.
FIG. 4
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.
FIG. 5
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.
FIG. 6
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.
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