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1 Department of Developmental Biology (VIB-07), Flanders Interuniversity Institute for Biotechnology (VIB), and Laboratory of Molecular Biology (Celgen), University of Leuven, B-3000 Leuven, Belgium
2 Department of Cell Biology, Biozentrum, University of Basel, CH-4056, Switzerland
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Abstract |
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Introduction |
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In a few cases TGF-ß signaling is known to result in the regulation of gene expression at the post-transcriptional level also. This usually occurs through modulation of mRNA turnover. For example, TGF-ß1 stimulates tropoelastin production in adult rat fibroblasts by increasing the stability of its mRNA. This occurs through a reduction of the binding activity of a cytosolic 50 kDa protein to a specific element within the tropoelastin mRNA open reading frame (Zhang et al. 1999). Tropoelastin mRNA levels decrease as the binding activity of the 50 kDa protein increases. TGF-ß1 regulates also the stability of Indian hedgehog (Ihh) (Murakami et al. 1997) and Pax2 mRNAs (Liu et al. 1997) but the precise mechanisms remain to be elucidated. Indeed, post-transcriptional regulation of gene expression by members of the TGF-ß family remains altogether a largely unexplored field.
We report the cloning and characterization of the novel Smad-interacting protein Smicl. It is related to the 30 kDa subunit of the pre-mRNA processing factor Cleavage and Polyadenylation Specificity Factor (CPSF). CPSF is one of the six factors that act together to achieve efficient pre-mRNA processing in vitro. Functional CPSF consists of at least four subunits (160, 100, 73 and 30 kDa, respectively) and participates in both cleavage and polyadenylation of de novo transcripts. Smicl was found to interact with a number of these CPSF subunits. Moreove, Smicl dependent induction of a TGF-ß responsive reporter gene requires the presence of an intact cleavage/polyadenylation signal. Thus, Smicl is a new CPSF interacting protein and component TGF-ß signaling cascades. The described interactions may point to the existence of mechanisms coupling Smad dependent transcription with pre-mRNA processing.
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Results |
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A yeast two-hybrid screening was carried out with the Smad1 MH2 domain as bait and cDNA prepared from mouse E12.5 embryos as source of prey polypeptides (Verschueren et al. 1999). In addition to SIP1, which is a DNA-binding transcriptional repressor (Remacle et al. 1999; Comijn et al. 2001; Tylzanowski et al. 2001), we identified another polypeptide (named th12) of 352 amino acids (aa) in-frame with the GAL4-transactivation domain of the prey plasmid. Cloning and sequencing of the complete open reading frame (950 amino acids) revealed that the th12 polypeptide encompasses aa 2–353 of the full-length protein. Whereas this polypeptide interacted with the mouse Smad2 and Smad5 MH2 domains, it did not interact with a Smad1 MH2 domain from which the last 40 amino acids were removed, suggesting that this part of R-Smads is essential for interaction. In addition, the GST fusion proteins containing the Smad1, Smad2 or Smad5 MH2 domain specifically pulled down th12 from crude HeLa cell extracts (Fig. 1, lanes 6, 2 and 4, respectively), whereas no pull-down was observed with the negative controls (Fig. 1, lanes 7–10 and 1, 3, 5). This confirms the specificity of the th12/Smad interaction.
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The th12 cDNA encoded a continuous open reading frame that lacked a translation start or stop codon. However, it displayed a high degree of sequence identity over a region of 1000 nucleotides with (the partial) human KIAA0150 cDNA. Based on this and other sequence homologies found in the EST database, standard PCR techniques were used to obtain a full-length cDNA encoding a protein of 950 amino acids (accession number AJ516034). A striking feature of this protein is the presence of a zinc finger cluster containing five fingers of the CCCH-type (Fig. 2A), which is a motif found in the RNA-binding protein CPSF-30 (accession number AF033201; Fig. 2B). Consequently, the novel protein was named Smad-interacting CPSF-like factor (Smicl).
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To investigate the function of Smicl in TGF-ß signaling, we first documented the interaction between Smicl and Smads in mammalian cells. A plasmid encoding Myc-tagged Smicl (MycSmicl) was co-transfected with plasmids encoding Flag-tagged Smad1, Smad3 or Smad4 proteins, with or without constitutively active (ca) ALK6 (a type I BMP receptor known to activate Smad1 (Hoodless et al. 1996; Kretzschmar et al. 1997)) or caALK4 (for obtaining Smad3 activation (Nakao et al. 1997)). Smad proteins were immunoprecipitated with an anti-Flag antibody and the precipitates were analyzed by Western blotting using anti-Myc antibody. Weak binding of transfected Smad1 or Smad3 to Smicl was detectable in non-stimulated cells (Fig. 3A, lanes 5 and 3, respectively) but this binding increased strongly upon co-transfection of the active variant of the respective type I receptors (Fig. 3A, lanes 4 and 2). The binding of Smicl to Smad4 was also stimulated by activation of the Smad signal transduction pathway (Fig. 3A, lanes 6 and 7). The same results were obtained when Smicl was precipitated first, followed by detection of the Smad proteins (data not shown).
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Smicl potentiates ALK4 induced Smad dependent transcriptional responses
R-Smads and Smad4 bind to DNA and activate transcription after stimulation of cells with TGF-ß/activins or BMPs (Massague & Wotton 2000). Accordingly, transcription of a synthetic reporter gene, containing in its regulatory region four copies of a Smad-binding sequence (4xSBE), derived from the JunB promoter, was induced upon co-expression of caALK4 or caALK6, as was reported before (Jonk et al. 1998) (Fig. 4A,B). Importantly, co-expression of Smicl enhanced the caALK4 mediated induction significantly (Fig. 4A), whereas the transcriptional response induced by caALK6 was not affected by Smicl (Fig. 4B). From the latter, we can conclude that although activated Smad1, acting downstream of ALK6 was found to bind Smicl in co-immunoprecipitation assays, this interaction appears to be non-functional, at least in the conditions tested.
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SBD), which was mapped by a yeast two-hybrid assay to amino acids 35–89 (data not shown) was generated. This mutant variant did not detectably interact with R-Smads in a co-immunoprecipitation (co-ip)/Western analysis (see Fig. 6C). Nevertheless, SmiclSBD was produced at levels comparable to wild-type Smicl and, similar to the latter, was present in the nucleus (data not shown). This mutant Smicl protein was much less potent than wild-type Smicl in enhancing the caALK4 mediated induction of reporter activity, although a residual activity was still seen (Fig. 4A). This residual activity could be due to remaining interaction with Smads below the detection limit of our co-ip/Western assay. Alternatively, there might be some residual indirect interaction via other proteins, since Smads and Smicl may be part of bigger complexes.
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Sequence similarity and features of the Smicl 5xCCCH domain
Smicl contains five zinc fingers in its C-terminal portion (Fig. 2A), and a similar 5xCCCH domain was found in the C-terminal part of mammalian CPSF-30 (accession numbers AF033201 and BTU96448) and in CPSF-30 homologs such as Drosophila clipper (Bai & Tolias 1996, 1998). CPSF-30 is an RNA-binding protein required for cleavage and polyadenylation of pre-mRNA (Barabino et al. 1997, 2000). The zinc finger domain of clipper has been shown to bind in a nonspecific manner to single-stranded DNA (ssDNA) but not to double-stranded (ds) DNA, and to possess ribonuclease (RNase) activity (Bai & Tolias 1996). Hence, we investigated whether the 5xCCCH domain of Smicl (amino acids 634–803 produced as a GST fusion protein) binds to ssDNA and degrades the same RNA substrate as the clipper zinc fingers. A gel retardation assay was carried out with single-stranded kappa immunoglobulin heavy chain enhancer oligonucleotide (
E2; Sekido et al. 1994) as a probe. As a control, the same oligonucleotide, but now in its double-stranded form, was used. The Smicl 5xCCCH domain was found to bind ssDNA but not dsDNA (Fig. 5A). As expected, the C-terminal zinc finger cluster of SIP1, previously shown to bind to the E2 probe (Verschueren et al. 1999), bound strongly to the dsDNA probe but not to the ssDNA probe.
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Smicl binds to CPSF subunits and Smad proteins affect binding of CPSF-100 to Smicl
The nuclear localization of Smicl, and the structural and functional similarities of its zinc finger domain with CPSF-30, suggested a possible association of Smicl with CPSF subunits. This was confirmed by co-immunoprecipitation experiments. Immunoprecipitates of endogenous Smicl were found to contain at least one of the endogenous CPSF subunits: CPSF 73 (Fig. 6A). This prompted us to verify whether Smads could be part of a Smicl-containing CPSF complex. Myc-tagged Smicl could be co-immunoprecipitated with endogenous CPSF-100 in these experiments (Fig. 6B, lane 1), but we could not detect endogenous Smad2 or Smad3 in this immunoprecipitate (Fig. 6B, lane 3). In contrast, endogenous Smad2/3 was found to interact with Myc-tagged Smicl (Fig. 6B, lane 4), even without co-transfection of an expression construct encoding caALK4. Most likely, this is due to the presence of a basal level of TGF-ß signaling in HEK293T cells, as seen by the presence of phosphorylated Smad2 without exogenous TGF-ß stimulation (data not shown and Verschueren et al. 1999).
In conclusion, it appeared that Smicl and Smads do not bind simultaneously to CPSF-100. We then asked whether this mutually exclusive binding could be due to competition of Smads and CPSF-100 for a common binding site in Smicl. Deletion of the Smad binding domain abolished interaction of Smicl with Smad3, but not with the 100 kDa subunit, indicating that the proteins interact through separate domains in Smicl (Fig. 6C). Thus, mutually exclusive interaction could occur by binding of one protein inhibiting the binding of the other.
The presence of a cleavage/polyadenylation signal on pre-mRNA is required for Smicl to potentiate caALK4-mediated induction of reporter activity
The observations that the Smicl 5xCCCH domain binds to ssDNA and has RNase activity, and that Smicl associates with CPSF subunits, raised the question of whether Smicl affects 3'-end processing of target pre-mRNAs. We therefore studied the requirement of the poly(A) signal for Smicl-dependent potentiation of TGF-ß/Smad reporter gene activity. To verify the involvement of this cis-acting sequence, we used the 3TP-Lux reporter plasmid, which can be linearized upstream of its SV40 early poly(A) signal. Such an approach leaves the luciferase sequence intact but should lead to premature termination of the transcript by a run-off event, as no other 3'-end processing signal is present in cis. This strategy was used rather than deletion of the sequence in the plasmid because this would yield transcriptional read-through resulting in no luciferase activity.
As observed in experiments using the 4xSBE reporter, Smicl potentiated the response of this reporter to caALK4 in CHO cells. Moreover, the activity of the linearized reporter was substantially lower than the activity of the circular reporter, but the relative induction by caALK4 occurred to the same extent for the linearized reporter compared to the intact one. However, co-transfection of Smicl could no longer potentiate the response of the linearized reporter (Fig. 7A). In contrast, a reporter that was linearized downstream of the polyadenylation signal was still responsive to Smicl (Fig. 7B). We conclude that the Smicl activity in this assay requires the presence of the intact (i.e. not cut away from the preceding coding sequence) 3'-end processing sequence. Moreover, a Smicl polypeptide (th12) lacking the zinc finger domain did not potentiate induction of the reporter, in contrast to full-length Smicl. Thus, the domain with CPSF30-like activity is also required for Smicl's activity in this assay, further suggesting a role for Smicl in 3'-end formation. Altogether, our data suggest a novel mechanism of Smad-dependent regulation of gene expression, possibly in conjunction with CPSF complexes.
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Discussion |
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Smicl contains a 5xCCCH domain in its C-terminal portion. A similar domain is present in CPSF-30. CPSF-30 is a CPSF subunit that together with CPSF-73, -100 and -160 is involved in cleavage and polyadenylation of pre-mRNA. CPSF-30 is a nuclear protein in mammalian somatic cells (Jenny et al. 1994). It can be cross-linked to an AAUAAA-containing RNA substrate (Jenny et al. 1994). The requirement for CPSF-30 in pre-mRNA cleavage and polyadenylation has been demonstrated in vitro, and the second zinc finger is required for this in vitro activity (Barabino et al. 1997, 2000). In addition, the 5xCCCH domain can bind to ssDNA and displays an RNase activity. Together, these data suggest that the activity of the zinc finger cluster in CPSF-30 is important for CPSF mediated cleavage and polyadenylation.
We have found that the 5xCCCH domain of Smicl possesses a similar activity to the zinc finger domain of CPSF-30 inasmuch as it can bind to ssDNA and cleave RNA. In accordance with this, Smicl was found here in a complex with at least two subunits of CPSF, i.e. CPSF-100 and CPSF-73. Thus, Smicl could be part of the 3' pre-mRNA processing machinery. Interaction of Smicl with activated Smad3 or CPSF subunits occurs through distinct domains but nevertheless is mutually exclusive. Smicl might thus be a bifunctional protein that can associate with CPSF subunits on the one hand or engage in a complex with activated and nuclear Smads increasing TGF-ß induced transcriptional responses on the other hand. Importantly, however, the activity of Smicl on potentiation of TGF-ß induced expression of the 3-TPLux reporter required the presence of a cleavage and polyadenylation sequence in cis. How can this observation be reconciled with the mutually exclusive binding of Smicl to either activated Smad3 or CPSF subunits? The answer could come from the fact that transcriptional initiation and 3'-end processing are mechanistically coupled processes. It was shown that CPSF is recruited to the transcription initiation site by TFIID, translocates to the elongation complex and is then transferred to the cleavage/polyadenylation sequence of pre-mRNA (Minvielle-Sebastia & Keller 1999). Similarly, Smicl could be recruited to the promoter of specific genes by transcription factors (such as Smad proteins) and then translocate to the CPSF complex to participate in 3'-end processing, leading to increased gene expression (Fig. 8).
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Experimental procedures |
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Cells (HEK293T, COS1 and HeLa) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) supplemented with 4.5 g glucose/L for HEK293T cells. Chinese Hamster Ovary (CHO) cells were grown in Ham's F12 with 10% FBS. Cells were transfected using Fugene (Roche Molecular Biochemicals) according to the manufacturer's protocol.
Yeast two-hybrid cloning and assays
For cloning of bait cDNA (for Smads) in pGBT9 and prey cDNAs in pAct2 (Matchmaker I and II, Clontech) see Verschueren et al. (1999). Yeast transformations (strain CG-1945) were performed according to Gietz et al. (1992).
GST-pull down assay
Th12, HA-tagged at its N-terminal end, was produced in HeLa cells with the T7 vaccinia virus (T7 vv) expression system as described in Wuytens et al. (1999). Smad1, Smad2 and Smad5 MH2 domains were produced in E. coli as fusion proteins with GST and used to pull-down th12 polypeptide from cell extracts as described in Verschueren et al. (1999).
Identification of the full-length Smicl cDNA sequence
The full-length mouse Smicl cDNA sequence was obtained by a combination of PCR and 5' RACE (5' RACE kit; Life Technologies) according to the manufacturer's protocol.
Co-immunoprecipitation experiments
cDNAs encoding Smicl or Smicl lacking the SBD (SmiclSBD) were subcloned in pCS3 (Rupp et al. 1994) resulting in an N-terminal fusion with the (Myc)6 tag. For construction of Flag-tagged Smads and caALK receptors, see Verschueren et al. (1999). Polyclonal anti-peptide antiserum was raised in rabbits against a mixture of two Smicl peptides (QAPRSPRTKDSGKPL and KAPPGTPRWRNKGYR). Extracts of HEK293T cells were subjected to immunoprecipitations as described in Verschueren et al. (1999). Endogenous CPSF-100, CPSF-73 and Smicl were visualized using anti-CPSF100 (Jenny et al. 1994), anti-CPSF73 (Jenny et al. 1996) and the anti-Smicl polyclonal antisera in combination with HRP-conjugated anti-rabbit antibody (Jackson). Endogenous Smad 2/3 was visualized using anti-Smad 2/3 ((N-19) X; Santa Cruz Biotechnology) and HRP-conjugated anti-goat antibody (Jackson) and the enhanced chemiluminiscence kit (New England Nuclear).
Immunofluorescence
Indirect immunofluorescence was done in transfected COS cells grown on chamber slides, about 24 h after their transfection according to Meersseman et al. (1997). The same protocol was applied for subcellular localization of endogenous Smicl in HeLa cells. Purified Smicl anti-peptide antibody (Eurogentec) and cy2-conjugated goat anti-rabbit IgG were used as primary and secondary antibodies, respectively.
Production of GST fusion proteins
The Smicl 5xCCCH zinc finger domain was obtained as a GST fusion protein by cloning the Smicl cDNA sequence encoding amino acids 634–803 in pGEX-4T-3 (Amersham Pharmacia Biotech). GST fusion proteins of Smicl and of CD40 (Pype et al. 2000), PLAG (Kas et al. 1998), th1 and SIP1CZF (Verschueren et al. 1999) were expressed in E. coli strain BL21. Bacterial cultures were lyzed in BPER reagent (Sigma) and proteins were purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech). Proteins were eluted from the beads with GST elution buffer, containing 0.5 mM ZnSO4, and glycerol was added to a final concentration of 10% for storage at –80 °C. Protein concentrations were estimated on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels (PAG).
Electrophoretic mobility assay
The E2 (Sekido et al. 1994) oligonucleotides were labeled with [
32P]-ATP and T4 polynucleotide kinase (New England Biolabs). GST-fusion proteins were prepared from E. coli (see above) and the DNA binding assay was performed as described in Remacle et al. (1999).
Identity and preparation of the RNA probe
Substrate I (Bai & Tolias 1996) was cloned as an EcoRI/HindIII DNA fragment in pGEM3Z (Promega Corp., Madison, WI) downstream from the T7 promoter. A radio-labeled RNA probe was prepared by in vitro transcription using T7 RNA polymerase. DNA was degraded by RNase-free DNase, and the probe was purified on a Bio-Spin 30 chromatography column (Biorad).
RNA degradation assay
Cleavage was performed in 50 µL reaction mixtures in binding buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 7 mM dithiothreitol) and contained ±250 ng labeled RNA and ±90 ng GST-coupled proteins, as well as 2 µL of RNasin (40 U/µL). At different time-points, 10 µL samples were collected and the degradation reaction was stopped by adding it to 10 µL of STOP solution (90% formaldehyde and 0.1% bromophenol blue), immediately frozen, and later separated on a denaturing 12% PAG, 7 M urea.
Reporter assays
For the 4xSBE and 3TP-Lux assays, HEK293T and CHO cells, respectively, were collected 30–48 h after transfection. Transfections were done in triplicate in 24-well plates. Each well was transfected with 450 ng of plasmid DNA, 50 ng of luciferase reporter, 50 ng of CMV promoter-based lacZ reporter, and combinations of 50 ng of c.-receptor constructs (caALK4 or caALK6), and 300 ng of N-terminally (Myc)6-tagged Smicl, SmiclSBD cDNA or th12 in pCS3. The total amount of DNA was kept constant by adding empty pGEM vector. Cell extracts were assayed for luciferase activity and ß-galactosidase activity according to the manufacturers’ protocols (Promega (Madison, WI, USA) and Clontech (Palo Alto, CA, USA), respectively). The 3TP-Lux reporter construct was linearized in front of and behind its cleavage and polyadenylation sequence with Pfml I and AccI, respectively, and subsequently purified after agarose gel electrophoresis. The data were normalized by calculating the ratio of the luciferase to ß-galactosidase activities. The average normalized luciferase activity is presented relative to the activity in non-stimulated samples as x-fold activation.
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Acknowledgements |
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Footnotes |
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Present addresses: aDipartimento di Biotechnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, Milano, Italy;
bJanssen Research Foundation, Turnhoutsesteenweg 30, B-2340 Beerse, Belgium.
* Correspondence: E-mail: kristin{at}med.kuleuven.ac.be |
References |
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Received: 25 February 2005
Accepted: 13 June 2005
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100) and controlled in Western blots (W) with a polyclonal anti-CPSF100 antiserum (lane 1). Co-immunoprecipitation of MycSmicl or endogenous Smad2/3 was investigated by immunoblotting of the precipitate with anti-Myc (lane 1) or polyclonal anti-Smad2/3 antibodies (lane 3). To analyze binding of endogenous Smad2/3 with Myc-tagged Smicl, anti-Myc immunoprecipitates were analyzed for the presence of these proteins by immunoblotting using the polyclonal anti Smad2/3 antibodies (lane 4). Lanes 2 of 
