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Cloning and characterization of a novel RNA polymerase II C-terminal domain phosphatase
BBRC Biochemical and Biophysical Research Communications 331 (2005) 1401–1407 www.elsevier.com/locate/ybbrc
Cloning and characterization of a novel RNA polymerase II C-terminal domain phosphatase Huarui Zheng, Chaoneng Ji, Shaohua Gu, Binying Shi, Jin Wang, Yi Xie, Yumin Mao * State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PeopleÕs Republic of China Received 11 April 2005 Available online 22 April 2005
Abstract Reversible phosphorylation of RNA polymerase (RNAP) IIÕs largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. In this study, we isolated a novel phosphatase gene by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA is 2215 bp in length, encoding a 318-amino acid polypeptide that contains a ubiquitin-like domain and a CTD phosphatase domain. Therefore, it was termed ubiquitin-like domain containing CTD phosphatase 1 (UBLCP1). Reverse transcription PCR (RT-PCR) revealed that UBLCP1 was expressed with relatively lower levels in most adult normal tissues and higher levels in fast growing or tumor tissues. Transient transfection experiment suggested that UBLCP1 was localized in the nucleus of COS-7 cells. Significantly, UBLCP1 could dephosphorylate GST-CTD in vitro. Accordingly, UBLCP1 may play a role in the regulation of phosphorylation state of RNA polymerase II C-terminal domain. 2005 Elsevier Inc. All rights reserved. Keywords: cDNA; CTD phosphatase; Expression pattern; Proteasome; RNA polymerase II; Transcription
The largest subunit of RNA polymerase (RNAP) II contains a C-terminal domain (CTD) composed of a tandemly repeated heptapeptide motif (consensus sequence YSPTSPS). Reversible phosphorylation of CTD plays a key role in the progression of RNAP II through the transcription cycle [1]. The hypophosphorylated form of RNAPII, designated RNAPIIA, was demonstrated to be essential for preinitiation complex formation and transcription initiation, whereas hyperphosphorylated RNAPII, designated RNAPIIO, is involved in transcription elongation [2,3]. Furthermore, the level and pattern of CTD phosphorylation also affect pre-mRNA processing events, including capping, splicing, and 3 0 end processing [4,5]. The dynamic phosphorylation state of the CTD reflects a kinetic balance
*
Corresponding author. Fax: +86 21 65642502. E-mail address: [email protected] (Y. Mao).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.04.065
between the multiple CTD kinases and CTD phosphatase activities found in eukaryotic cells. A large variety of kinases have been reported to phosphorylate CTD in vitro [6]. In contrast, few CTD phosphatases have been identified. Most studies were focused on FCP1 [7–13], the first validated CTD phosphatase. FCP1 is a class C phosphatase containing a BRCT domain that is required for interaction with RNAPII and dephosphorylation of the CTD. FCP1 orthologs are present in all known eukaryotic organisms and the enzyme is essential for cell viability in budding and fission yeast [7,12]. Mammalian FCP1 dephosphorylates RNAP II Ser-2 and Ser-5 with comparable efficiencies in vitro [14]. However, yeast FCP1 appears to be more specific for Ser-2 when synthetic peptide served as the substrate [10]. Not long ago, a small FCP1 related protein, SCP1, has been identified as being CTD phosphatase with Ser-5 preference. SCP1 also contains a phosphatase domain similar to that of FCP1 [15].
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Recently, the yeast Ssu72 [16], as well as CPL1 and CPL2 in Arabidopsis [17], are also demonstrated to be CTD phosphatases with specificity for Ser-5. Given the importance of CTD phosphorylation in gene expression, it is reasonable to speculate that there should exist additional CTD phosphatases. During a large sequencing analysis of a human fetal brain cDNA library, we isolated a gene that contains a catalytic domain with homology to the CTD phosphatase domain of FCP1 and SCP1. In the present study, we showed its phosphatase activity toward GST-CTD in vitro and possible preference for Ser-5 within the CTD consensus repeat. Materials and methods Cloning of UBLCP1 gene. The cDNA of UBLCP1 gene was cloned from a human fetal library during large-scale cDNA sequencing. Human fetal brain poly(A) RNA was purchased from Clontech. Double-stranded cDNAs were prepared with the SMART cDNA Library Construction Kit (Clontech). The pBluescript II SK (+) vector (Stratagene) with a modified MCS was used to clone the cDNAs. The cDNA inserts were sequenced on an ABI PRISM 377 DNA sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Kit and BigDye Primer Cycle Sequencing Kit (Perkin-Elmer) with a -21M13 primer, a M13Rev primer, and synthetic internal walking primers designed according to the cDNA sequence fragments. Each part of the insert was sequenced at least three times bi-directionally. Subsequent editing and assembly of all the sequences from one clone was performed using Acembly (SangerÕs Center). Sequence analysis. BLAST-N searching against the human genome was performed to identify the chromosomal localization of the UBLCP1 gene. cDNA and deduced amino acid sequence comparisons were carried out using BLAST-N and BLAST-P at NCBI (http://www. ncbi.nlm.nih.gov/blast). ClustalW (http://www.ebi.ac.uk/clustalw/ index.html) and GeneDoc were used to perform multiple sequence alignment. Tissue expression pattern of UBLCP1. Human Multiple Tissue cDNA and tumor tissue cDNA panels were purchased from Clontech. The sequences of UBLCP1 specific primers were: 5 0 TTC TAA ATT CTG AGC GGT CTC AGT T 3 0 (UBLCP1 F) and 5 0 ACA CCA CTG AAA TTA GTC CTG TAA G 3 0 (UBLCP1 R). G3PDH control primers were 5 0 -TGA AGG TCG GAG TCA ACG GAT TTG GT-3 0 (G3PDH F) and 5 0 -CAT GTG GGC CAT GAG GTC CAC CAC-3 0 (G3PDH R). Amplification (30 s at 94 C, 45 s at 56 C, and 1.0 min at 72 C) of 36 cycles (for UBLCP1) and 24 cycles (for G3PDH) was performed using ELONGASE DNA polymerase (Gibco-BRL). The PCR products of UBLCP1 and G3PDH were then visualized on a 1.2% agarose gel. Subcellular localization of UBLCP1. COS-7 cells were cultured in DulbeccoÕs modified EagleÕs medium containing 10% fetal bovine serum. The cells were split on 35 mm dishes at 1 · 106 per dish. After 24 h, the cells were transiently transfected with pEGFP-UBLCP1 by using LipofectAMINE 2000 (Invitrogen) according to the manufacturerÕs protocol. After 48 h of transfection, cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and then washed with PBS three times. The cells were stained with DAPI for 10 min and washed with Milli-Q. The coverslips were mounted and viewed using a Leica fluorescence microscope. Expression in bacteria and purification of GST-UBLCP1 and GSTCTD. The ORF of UBLCP1 was amplified and digested with BamHI and EcoRI. The product was subsequently cloned into the pGEX-4T1 expression vector (Pharmacia). Transformants of Escherichia coli
BL21 with the resulting plasmid constructs were grown in 400 ml LB medium containing 100 lg/ml ampicillin until the absorbance at 600 nm reached 0.6–0.8. IPTG was added to a final concentration of 0.4 mM, and then the culture was induced at 28 C for 3 h. Protein was purified from soluble bacterial lysates with glutathione–Sepharose 4B affinity chromatography according to the manufacturerÕs protocol and its concentration was determined by the method of Bradford using BSA as a standard [18]. SDS–PAGE was performed to check the integrity of the fusion protein. GST-CTD was expressed and purified as described previously [19]. Phosphatase assay towards pNPP. Reaction mixtures (1 ml) containing 50 mM Tris acetate (5.0), 10 mM MgCl2, 20 mM p-nitrophenyl phosphate (pNPP), and GST-UBLCP1 or GST alone as specified were incubated at 37 C for 1 h. Then the reactions were quenched by adding 1 ml of 2 M sodium carbonate. The formation of p-nitrophenyl was monitored by measuring the absorbance at 420 nm on an Ultraspec 4000 spectrophotometer (Pharmacia). CTD phosphatase assay. GST-CTD substrate was prepared by phosphorylation with Cdc2 kinase (New England Biolabs) for 12 h at 30 C in kinase buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, and 0.01% Brij 35), by adding 4 mM ATP. CTD phosphatase assays were carried out in the buffer of 50 mM Tris acetate, 10 mM MgCl2, GST-CTDo and purified GST-UBLCP1, as described previously [17], and the pH of assay mixes were verified to be near 7.0. Reactions were incubated for 3 h at 37 C, stopped by adding SDS loading buffer. The Ser-2 and Ser-5 phosphorylation state was analyzed by immunoblotting with H5 and H14 monoclonal antibodies (Covance), respectively. Briefly, samples were subjected to 10% SDS–PAGE followed by blotting onto PVDF membrane (Millipore). The membrane was blocked in 10% skim milk for 1 h at room temperature, then incubated with H5 or H14 monoclonal antibodies (diluted 1:500) overnight, and followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse Ig M secondary antibody (diluted 1:2000; Santa Cruz) for 1 h at room temperature. Finally, the membrane was developed using an enhanced chemiluminescence (ECL) detection kit (Pierce).
Results and discussion Identification of human UBLCP1 Examination of database reveals a cDNA sequence of UBLCP1 (Accession No. AK057996) which is identical (with the exception of about 20 base deletion both in 5 0 and 3 0 -end) to the sequence we cloned. But its function has not been investigated yet. Our sequence has also been submitted to GenBank under Accession No. AY444562. The cDNA has 2215 bp and contains a large open reading frame from nucleotide 139 to 1095, encoding a protein with 318 amino acid residues. The result of BLAST-N search against human genome database of GenBank shows that the gene is located on 5q33.3. It spans more than 22.7 kb of genomic DNA and consists of 11 exons. The sequence around the ATG start codon (AGAATGG) conforms to Kozak consensus. There is an upstream in-frame stop codon at position 106 and three polyadenylation signals near 3 0 end. The predicted molecular weight of this protein is 36.8 kDa and the isoelectric point is 6.27. BLAST-P search result revealed that UBLCP1 has a catalytic domain of CTD phosphatase (CPDc) from residue 136 to 271, which also exists in FCP1 and SCP1
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Fig. 1. Sequence alignments of UBLCP1 to related CTD phosphatases and ubiquitin-like proteins. (A) Schematic domain organization of human FCP1, SCP1, and UBLCP1. (B) Alignment of the catalytic domains from human FCP1, SCP1, and UBLCP1. The bracket indicates the conserved signature motif. The active site residues are marked with an asterisk below the alignment. The numbers in the right indicate the position of amino acid in the full length sequence. (C) Alignment of the UBL domain of UBLCP1 and several PIM-containing UBLPs. The sequences are denoted by their gene name followed by the species abbreviation and GenBank identifier [23]. The five residues comprising PIM are shaded.
(Fig. 1A). A short conserved motif (143DVDYT147) located near the N-terminal margin of the catalytic domain corresponds to the signature sequence DXDX(T/V) in FCP1 and SCP1 (Fig. 1B), which is essential for their phosphatase activities. Recently, the crystal structure of the catalytic domain of SCP1 has been resolved and the catalytic mechanism has been demonstrated. The first aspartate in the signature motif undergoes metal-assisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate [20]. Furthermore, SCP1 structure shows that seven residues, including three residues in the signature motif, form the active center (D96, D98, T100, T152, K190, D206, and N207 in SCP1 and D188, D190, T192, T246, R288, D302, and D303 in FCP1). All these essential residues also exist in UBLCP1 with corresponding positions (D143, D145, T147, S183, K230, D252, and D253) (Fig. 1B), which are conserved in all known UBLCP1
orthologs (data not shown). Although the overall sequence identity in the catalytic domains of human FCP1, SCP1, and UBLCP1 is low, the conservation of these essential residues is significant. These data strongly suggest that UBLCP1 is structurally and mechanistically related with FCP1 and SCP1. Interestingly, the UBLCP1 protein contains a ubiquitin-like (UBL) domain at N-terminal, which is absent in FCP1 and SCP1 (Fig. 1A). Ubiquitin has a well-documented role in targeting protein for degradation by the proteasome, but additional effects are recently being uncovered at a rapid rate. Especially, the genomes of yeast and higher eukaryotes encode numerous UBL domain-containing proteins (UBLPs), which appear to be involved in a diverse series of cellular functions such as DNA repair, autophagy, and signal transduction [21,22]. However, recent sequence analyses of UBLPs revealed that a group of these proteins contain a protea-
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some-interacting motif (PIM), which consists of five residues [23]. The UBL domain of UBLCP1 was compared with several of these UBLPs. The result indicated that at least four of the PIM residues also exist in UBLCP1 (Fig. 1C) and is likely to be indicative of the ability of UBLCP1 to interact with the proteasome.
Database search result shows that UBLCP1 orthologs are present in many organisms, ranging from Mus musculus to Dictyostelium discoideum. The conservation and structural characteristic of UBLCP1 suggest that it may play an important role in living organisms. Expression pattern of UBLCP1 gene The tissue distribution of UBLCP1 mRNA was examined by RT-PCR using multiple tissue cDNA and tumor tissue cDNA panels (Clontech) as templates. Expression of UBLCP1 mRNA was detected with relatively higher level in placenta, lung, testis, and ovary. The expression level was relatively weak in heart, liver, kidney, spleen, thymus, colon, and peripheral blood leukocyte. Expression of UBLCP1 mRNA nearly cannot be detected in other tissues examined (Fig. 2A). Strikingly, the expression level of UBLCP1 was particularly high in most tumor tissues (Fig. 2B). On the whole, compared with those of normal tissues, the expression levels of UBLCP1 in tumor tissues were upregulated obviously. These results suggest that UBLCP1 may be involved in the process of tumorigenesis. Subcellular localization of UBLCP1
Fig. 2. The multiple tissue expression pattern of human UBLCP1 mRNA. Reverse transcription-PCR analysis of human normal adult cDNA and tumor tissue cDNA for UBLCP1 and G3PDH.
Subcellular localization of UBLCP1 was analyzed by transient transfection of EGFP-tagged UBLCP1. Although UBLCP1 lacks an obvious nuclear localization signal sequence, fluorescence observation and staining with DAPI for nuclear identification confirmed the specific localization of UBLCP1 in nucleus (Fig. 3). AD-293 cells were also transfected with pEGFP-UBLCP1 and the result (not shown) was the same as that in COS-7.
Fig. 3. Subcellular localization of UBLCP1. COS-7 cells transfected with pEGFP-UBLCP1 were stained with DAPI after fixation and observed under a fluorescence microscope (A–C). pEGFP was also transfected as a control (D–F).
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Expression and purification of UBLCP1
Fig. 4. SDS–PAGE analysis of expression and purification of GSTUBLCP1. Lane 1, total cell protein sample before induction of IPTG; lane 2, total cell protein sample after induction of 0.4 mM IPTG for 3 h; lane 3, soluble fraction of the cell lysate; and lane 4, flowthrough fraction. Lanes 5–7, GST-UBLCP1 protein after purification by glutathione–Sepharose 4B affinity chromatography. The positions and sizes (in kDa) of marker proteins are indicated on the left (lane M).
A prominent 62-kDa polypeptide was detected by SDS–PAGE in whole cell extracts of IPTG induced bacteria grown at 28 C. After centrifugal separation of the crude lysate, the GST-UBLCP1 was present predominantly in the soluble fraction. The fusion proteins were purified from the sonication supernatant of bacterial lysate by glutathione–Sepharose 4B affinity chromatography and analyzed by 10% SDS–PAGE. One major protein band corresponding to the expected 62 kDa recombinant protein was detected in elution fractions (Fig. 4).
Fig. 5. Enzymatic properties of UBLCP1. (A) Reaction mixtures containing 50 mM Tris acetate (pH 5.0), 20 mM pNPP, 10 mM MgCl2, and GSTUBLCP1 proteins as specified were incubated for 1 h at 37 C. (B) Effect of pH on the activity of UBLCP1. Reaction mixtures containing 50 mM Tris acetate (pH 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5), 10 mM MgCl2, 20 mM pNPP, and 10 lg UBLCP1 were incubated for 1 h at 37 C. (C) Effect of divalent cations on UBLCP1 activity. Reaction mixtures containing 50 mM Tris acetate (pH 5.0), 20 mM pNPP, 10 lg UBLCP1, and 10 mM each of the divalent cations.
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UBLCP1 is a CTD phosphatase with possible preference for Ser-5 GST-UBLCP1 fusion protein showed phosphatase activity toward p-nitrophenyl phosphate. The p-nitrophenyl reaction product was detected via its absorbance at 420 nm, which was directly proportional to the concentration of the recombinant protein (Fig. 5A). As reported for FCP1 from Schizosaccharomyces pombe and SCP1 utilizing pNPP as substrate [10,15], the optimal pH for UBLCP1 enzymatic activity is also near 5.0 (Fig. 5B). When using synthetic CTD peptides as substrate, the optimal pH for S. pombe FCP1 was also the same [10]. This unusual property might be ascribed to the catalytic mechanism of DXDX (T/V) family of phosphotransferases, which calls for an unprotonated aspartate nucleophile and a protonated aspartate general acid catalyst [20,24]. The phosphatase activity of UBLCP1 was Mg2+ dependent and Ca2+ could not substitute for Mg2+. Mn2+ also showed effect on increasing the activity, whereas Cu2+ and Zn2+ showed little effects on the activity (Fig. 5C). To determine whether UBLCP1 has CTD phosphatase activity and whether it displays any preference for Ser-2 or Ser-5, we assayed purified GST-UBLCP1 using a GST-CTD substrate, which had been phosphorylated using Cdc2 protein kinase. The GST-CTDo was detected by Western blot using H5 or H14 antibody. The results showed that UBLCP1 could effectively dephosphorylate Ser-5 site within CTD repeat. However, it seems UBLCP1 has relatively low activity on Ser-2 site (Fig. 6). Thus, these results indicated that UBLCP1 is a CTD phosphatase with possible preference for Ser-5 in vitro. Although its CTD phosphatase activity against RNAPII has not been validated in vivo, it is reasonable to speculate that UBLCP1 may play a role in regulating the phosphorylation state of RNAP II. During the transcription cycle, Ser-5 phosphorylation of RNAP II CTD is mainly detected at the promoter region, whereas Ser-2 phosphorylation is present in coding regions [25]. Given the possible preference of UBLCP1 for Ser-5, UBLCP1 is a candidate for acting early in the transcription cycle.
Fig. 6. UBLCP1 dephosphorylates GST-CTD. The GST-CTD was phosphorylated at both sites of Ser-2 and Ser-5 by Cdc2 kinase. The indicated amounts of GST-UBLCP1 or GST alone were added to each reaction. Phosphorylation status of CTD was detected by immunoblotting with H14 and H5, which is specific to Ser-5-PO4 and Ser-2-PO4, respectively.
Different from those CTD phosphatases previously identified, UBLCP1 has a ubiquitin-like domain in N-terminal, which contains a proteasome-interacting motif, indicating its potential ability of interacting with proteasome. In fact, our unpublished data showed that UBLCP1 could interact with a subunit of proteasome in vitro. Recent studies have revealed a number of connections between RNA polymerase II transcription and the proteasome [26,27]. Especially, it has been shown that the 26S proteasome and RNAP II can interact physically and functionally in vivo [28]. For the present, we cannot surmise the function of UBLCP1 with confidence, but considering the combination of the potential abilities of UBLCP1 to regulate the phosphorylation status of RNA polymerase II and to interact with the proteasome, we cautiously speculate that UBLCP1 may function in a certain period of transcriptional process, in which the proteasome might be involved.
Acknowledgments We thank Dr. William Dynan for the kind gift of the GST-CTD plasmid. We also thank Dr. Michael E. Dahmus for beneficial communication. This work was supported by a grant (10490193) from the Major Programs of the National Natural Science Foundation of China.
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[19] S.R. Peterson, A. Dvir, C.W. Anderson, W.S. Dynan, DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats, Genes Dev. 6 (1992) 426–438. [20] T. Kamenski, S. Heilmeier, A. Meinhart, P. Cramer, Structure and mechanism of RNA polymerase II CTD phosphatases, Mol. Cell 15 (2004) 399–407. [21] D.C. Schwartz, M.A. Hochstrasser, Superfamily of protein tags: ubiquitin, SUMO and related modifiers, Trends Biochem. Sci. 28 (2003) 321–328. [22] R. Hartmann-Petersen, C. Gordon, Integral UBL domain proteins: a family of proteasome interacting proteins, Semin. Cell Dev. Biol. 15 (2004) 247–259. [23] S.C. Upadhya, A.N. Hegde, A potential proteasome-interacting motif within the ubiquitin-like domain of parkin and other proteins, Trends Biochem. Sci. 28 (2003) 280–283. [24] S. Hausmann, H. Erdjument-Bromage, S. Shuman, Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: distributive mechanism, minimal CTD substrate, and active site mapping, J. Biol. Chem. 279 (2004) 10892–10900. [25] P. Komarnitsky, E.J. Cho, S. Buratowski, Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription, Genes Dev. 14 (2000) 2452–2460. [26] M. Muratani, W.P. Tansey, How the ubiquitin–proteasome system controls transcription, Nat. Rev. Mol. Cell Biol. 4 (2003) 192–201. [27] F.J. Herrera, S.J. Triezenberg, Molecular biology: what ubiquitin can do for transcription, Curr. Biol. 14 (2004) R622–R624. [28] T.G. Gillette, F. Gonzalez, A. Delahodde, S.A. Johnston, T. Kodadek, Physical and functional association of RNA polymerase II and the proteasome, Proc. Natl. Acad. Sci. USA 101 (2004) 5904–5909.
Cloning and characterization of a novel RNA polymerase II C-terminal domain phosphatase Huarui Zheng, Chaoneng Ji, Shaohua Gu, Binying Shi, Jin Wang, Yi Xie, Yumin Mao * State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PeopleÕs Republic of China Received 11 April 2005 Available online 22 April 2005
Abstract Reversible phosphorylation of RNA polymerase (RNAP) IIÕs largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. In this study, we isolated a novel phosphatase gene by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA is 2215 bp in length, encoding a 318-amino acid polypeptide that contains a ubiquitin-like domain and a CTD phosphatase domain. Therefore, it was termed ubiquitin-like domain containing CTD phosphatase 1 (UBLCP1). Reverse transcription PCR (RT-PCR) revealed that UBLCP1 was expressed with relatively lower levels in most adult normal tissues and higher levels in fast growing or tumor tissues. Transient transfection experiment suggested that UBLCP1 was localized in the nucleus of COS-7 cells. Significantly, UBLCP1 could dephosphorylate GST-CTD in vitro. Accordingly, UBLCP1 may play a role in the regulation of phosphorylation state of RNA polymerase II C-terminal domain. 2005 Elsevier Inc. All rights reserved. Keywords: cDNA; CTD phosphatase; Expression pattern; Proteasome; RNA polymerase II; Transcription
The largest subunit of RNA polymerase (RNAP) II contains a C-terminal domain (CTD) composed of a tandemly repeated heptapeptide motif (consensus sequence YSPTSPS). Reversible phosphorylation of CTD plays a key role in the progression of RNAP II through the transcription cycle [1]. The hypophosphorylated form of RNAPII, designated RNAPIIA, was demonstrated to be essential for preinitiation complex formation and transcription initiation, whereas hyperphosphorylated RNAPII, designated RNAPIIO, is involved in transcription elongation [2,3]. Furthermore, the level and pattern of CTD phosphorylation also affect pre-mRNA processing events, including capping, splicing, and 3 0 end processing [4,5]. The dynamic phosphorylation state of the CTD reflects a kinetic balance
*
Corresponding author. Fax: +86 21 65642502. E-mail address: [email protected] (Y. Mao).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.04.065
between the multiple CTD kinases and CTD phosphatase activities found in eukaryotic cells. A large variety of kinases have been reported to phosphorylate CTD in vitro [6]. In contrast, few CTD phosphatases have been identified. Most studies were focused on FCP1 [7–13], the first validated CTD phosphatase. FCP1 is a class C phosphatase containing a BRCT domain that is required for interaction with RNAPII and dephosphorylation of the CTD. FCP1 orthologs are present in all known eukaryotic organisms and the enzyme is essential for cell viability in budding and fission yeast [7,12]. Mammalian FCP1 dephosphorylates RNAP II Ser-2 and Ser-5 with comparable efficiencies in vitro [14]. However, yeast FCP1 appears to be more specific for Ser-2 when synthetic peptide served as the substrate [10]. Not long ago, a small FCP1 related protein, SCP1, has been identified as being CTD phosphatase with Ser-5 preference. SCP1 also contains a phosphatase domain similar to that of FCP1 [15].
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Recently, the yeast Ssu72 [16], as well as CPL1 and CPL2 in Arabidopsis [17], are also demonstrated to be CTD phosphatases with specificity for Ser-5. Given the importance of CTD phosphorylation in gene expression, it is reasonable to speculate that there should exist additional CTD phosphatases. During a large sequencing analysis of a human fetal brain cDNA library, we isolated a gene that contains a catalytic domain with homology to the CTD phosphatase domain of FCP1 and SCP1. In the present study, we showed its phosphatase activity toward GST-CTD in vitro and possible preference for Ser-5 within the CTD consensus repeat. Materials and methods Cloning of UBLCP1 gene. The cDNA of UBLCP1 gene was cloned from a human fetal library during large-scale cDNA sequencing. Human fetal brain poly(A) RNA was purchased from Clontech. Double-stranded cDNAs were prepared with the SMART cDNA Library Construction Kit (Clontech). The pBluescript II SK (+) vector (Stratagene) with a modified MCS was used to clone the cDNAs. The cDNA inserts were sequenced on an ABI PRISM 377 DNA sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Kit and BigDye Primer Cycle Sequencing Kit (Perkin-Elmer) with a -21M13 primer, a M13Rev primer, and synthetic internal walking primers designed according to the cDNA sequence fragments. Each part of the insert was sequenced at least three times bi-directionally. Subsequent editing and assembly of all the sequences from one clone was performed using Acembly (SangerÕs Center). Sequence analysis. BLAST-N searching against the human genome was performed to identify the chromosomal localization of the UBLCP1 gene. cDNA and deduced amino acid sequence comparisons were carried out using BLAST-N and BLAST-P at NCBI (http://www. ncbi.nlm.nih.gov/blast). ClustalW (http://www.ebi.ac.uk/clustalw/ index.html) and GeneDoc were used to perform multiple sequence alignment. Tissue expression pattern of UBLCP1. Human Multiple Tissue cDNA and tumor tissue cDNA panels were purchased from Clontech. The sequences of UBLCP1 specific primers were: 5 0 TTC TAA ATT CTG AGC GGT CTC AGT T 3 0 (UBLCP1 F) and 5 0 ACA CCA CTG AAA TTA GTC CTG TAA G 3 0 (UBLCP1 R). G3PDH control primers were 5 0 -TGA AGG TCG GAG TCA ACG GAT TTG GT-3 0 (G3PDH F) and 5 0 -CAT GTG GGC CAT GAG GTC CAC CAC-3 0 (G3PDH R). Amplification (30 s at 94 C, 45 s at 56 C, and 1.0 min at 72 C) of 36 cycles (for UBLCP1) and 24 cycles (for G3PDH) was performed using ELONGASE DNA polymerase (Gibco-BRL). The PCR products of UBLCP1 and G3PDH were then visualized on a 1.2% agarose gel. Subcellular localization of UBLCP1. COS-7 cells were cultured in DulbeccoÕs modified EagleÕs medium containing 10% fetal bovine serum. The cells were split on 35 mm dishes at 1 · 106 per dish. After 24 h, the cells were transiently transfected with pEGFP-UBLCP1 by using LipofectAMINE 2000 (Invitrogen) according to the manufacturerÕs protocol. After 48 h of transfection, cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and then washed with PBS three times. The cells were stained with DAPI for 10 min and washed with Milli-Q. The coverslips were mounted and viewed using a Leica fluorescence microscope. Expression in bacteria and purification of GST-UBLCP1 and GSTCTD. The ORF of UBLCP1 was amplified and digested with BamHI and EcoRI. The product was subsequently cloned into the pGEX-4T1 expression vector (Pharmacia). Transformants of Escherichia coli
BL21 with the resulting plasmid constructs were grown in 400 ml LB medium containing 100 lg/ml ampicillin until the absorbance at 600 nm reached 0.6–0.8. IPTG was added to a final concentration of 0.4 mM, and then the culture was induced at 28 C for 3 h. Protein was purified from soluble bacterial lysates with glutathione–Sepharose 4B affinity chromatography according to the manufacturerÕs protocol and its concentration was determined by the method of Bradford using BSA as a standard [18]. SDS–PAGE was performed to check the integrity of the fusion protein. GST-CTD was expressed and purified as described previously [19]. Phosphatase assay towards pNPP. Reaction mixtures (1 ml) containing 50 mM Tris acetate (5.0), 10 mM MgCl2, 20 mM p-nitrophenyl phosphate (pNPP), and GST-UBLCP1 or GST alone as specified were incubated at 37 C for 1 h. Then the reactions were quenched by adding 1 ml of 2 M sodium carbonate. The formation of p-nitrophenyl was monitored by measuring the absorbance at 420 nm on an Ultraspec 4000 spectrophotometer (Pharmacia). CTD phosphatase assay. GST-CTD substrate was prepared by phosphorylation with Cdc2 kinase (New England Biolabs) for 12 h at 30 C in kinase buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, and 0.01% Brij 35), by adding 4 mM ATP. CTD phosphatase assays were carried out in the buffer of 50 mM Tris acetate, 10 mM MgCl2, GST-CTDo and purified GST-UBLCP1, as described previously [17], and the pH of assay mixes were verified to be near 7.0. Reactions were incubated for 3 h at 37 C, stopped by adding SDS loading buffer. The Ser-2 and Ser-5 phosphorylation state was analyzed by immunoblotting with H5 and H14 monoclonal antibodies (Covance), respectively. Briefly, samples were subjected to 10% SDS–PAGE followed by blotting onto PVDF membrane (Millipore). The membrane was blocked in 10% skim milk for 1 h at room temperature, then incubated with H5 or H14 monoclonal antibodies (diluted 1:500) overnight, and followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse Ig M secondary antibody (diluted 1:2000; Santa Cruz) for 1 h at room temperature. Finally, the membrane was developed using an enhanced chemiluminescence (ECL) detection kit (Pierce).
Results and discussion Identification of human UBLCP1 Examination of database reveals a cDNA sequence of UBLCP1 (Accession No. AK057996) which is identical (with the exception of about 20 base deletion both in 5 0 and 3 0 -end) to the sequence we cloned. But its function has not been investigated yet. Our sequence has also been submitted to GenBank under Accession No. AY444562. The cDNA has 2215 bp and contains a large open reading frame from nucleotide 139 to 1095, encoding a protein with 318 amino acid residues. The result of BLAST-N search against human genome database of GenBank shows that the gene is located on 5q33.3. It spans more than 22.7 kb of genomic DNA and consists of 11 exons. The sequence around the ATG start codon (AGAATGG) conforms to Kozak consensus. There is an upstream in-frame stop codon at position 106 and three polyadenylation signals near 3 0 end. The predicted molecular weight of this protein is 36.8 kDa and the isoelectric point is 6.27. BLAST-P search result revealed that UBLCP1 has a catalytic domain of CTD phosphatase (CPDc) from residue 136 to 271, which also exists in FCP1 and SCP1
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Fig. 1. Sequence alignments of UBLCP1 to related CTD phosphatases and ubiquitin-like proteins. (A) Schematic domain organization of human FCP1, SCP1, and UBLCP1. (B) Alignment of the catalytic domains from human FCP1, SCP1, and UBLCP1. The bracket indicates the conserved signature motif. The active site residues are marked with an asterisk below the alignment. The numbers in the right indicate the position of amino acid in the full length sequence. (C) Alignment of the UBL domain of UBLCP1 and several PIM-containing UBLPs. The sequences are denoted by their gene name followed by the species abbreviation and GenBank identifier [23]. The five residues comprising PIM are shaded.
(Fig. 1A). A short conserved motif (143DVDYT147) located near the N-terminal margin of the catalytic domain corresponds to the signature sequence DXDX(T/V) in FCP1 and SCP1 (Fig. 1B), which is essential for their phosphatase activities. Recently, the crystal structure of the catalytic domain of SCP1 has been resolved and the catalytic mechanism has been demonstrated. The first aspartate in the signature motif undergoes metal-assisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate [20]. Furthermore, SCP1 structure shows that seven residues, including three residues in the signature motif, form the active center (D96, D98, T100, T152, K190, D206, and N207 in SCP1 and D188, D190, T192, T246, R288, D302, and D303 in FCP1). All these essential residues also exist in UBLCP1 with corresponding positions (D143, D145, T147, S183, K230, D252, and D253) (Fig. 1B), which are conserved in all known UBLCP1
orthologs (data not shown). Although the overall sequence identity in the catalytic domains of human FCP1, SCP1, and UBLCP1 is low, the conservation of these essential residues is significant. These data strongly suggest that UBLCP1 is structurally and mechanistically related with FCP1 and SCP1. Interestingly, the UBLCP1 protein contains a ubiquitin-like (UBL) domain at N-terminal, which is absent in FCP1 and SCP1 (Fig. 1A). Ubiquitin has a well-documented role in targeting protein for degradation by the proteasome, but additional effects are recently being uncovered at a rapid rate. Especially, the genomes of yeast and higher eukaryotes encode numerous UBL domain-containing proteins (UBLPs), which appear to be involved in a diverse series of cellular functions such as DNA repair, autophagy, and signal transduction [21,22]. However, recent sequence analyses of UBLPs revealed that a group of these proteins contain a protea-
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some-interacting motif (PIM), which consists of five residues [23]. The UBL domain of UBLCP1 was compared with several of these UBLPs. The result indicated that at least four of the PIM residues also exist in UBLCP1 (Fig. 1C) and is likely to be indicative of the ability of UBLCP1 to interact with the proteasome.
Database search result shows that UBLCP1 orthologs are present in many organisms, ranging from Mus musculus to Dictyostelium discoideum. The conservation and structural characteristic of UBLCP1 suggest that it may play an important role in living organisms. Expression pattern of UBLCP1 gene The tissue distribution of UBLCP1 mRNA was examined by RT-PCR using multiple tissue cDNA and tumor tissue cDNA panels (Clontech) as templates. Expression of UBLCP1 mRNA was detected with relatively higher level in placenta, lung, testis, and ovary. The expression level was relatively weak in heart, liver, kidney, spleen, thymus, colon, and peripheral blood leukocyte. Expression of UBLCP1 mRNA nearly cannot be detected in other tissues examined (Fig. 2A). Strikingly, the expression level of UBLCP1 was particularly high in most tumor tissues (Fig. 2B). On the whole, compared with those of normal tissues, the expression levels of UBLCP1 in tumor tissues were upregulated obviously. These results suggest that UBLCP1 may be involved in the process of tumorigenesis. Subcellular localization of UBLCP1
Fig. 2. The multiple tissue expression pattern of human UBLCP1 mRNA. Reverse transcription-PCR analysis of human normal adult cDNA and tumor tissue cDNA for UBLCP1 and G3PDH.
Subcellular localization of UBLCP1 was analyzed by transient transfection of EGFP-tagged UBLCP1. Although UBLCP1 lacks an obvious nuclear localization signal sequence, fluorescence observation and staining with DAPI for nuclear identification confirmed the specific localization of UBLCP1 in nucleus (Fig. 3). AD-293 cells were also transfected with pEGFP-UBLCP1 and the result (not shown) was the same as that in COS-7.
Fig. 3. Subcellular localization of UBLCP1. COS-7 cells transfected with pEGFP-UBLCP1 were stained with DAPI after fixation and observed under a fluorescence microscope (A–C). pEGFP was also transfected as a control (D–F).
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Expression and purification of UBLCP1
Fig. 4. SDS–PAGE analysis of expression and purification of GSTUBLCP1. Lane 1, total cell protein sample before induction of IPTG; lane 2, total cell protein sample after induction of 0.4 mM IPTG for 3 h; lane 3, soluble fraction of the cell lysate; and lane 4, flowthrough fraction. Lanes 5–7, GST-UBLCP1 protein after purification by glutathione–Sepharose 4B affinity chromatography. The positions and sizes (in kDa) of marker proteins are indicated on the left (lane M).
A prominent 62-kDa polypeptide was detected by SDS–PAGE in whole cell extracts of IPTG induced bacteria grown at 28 C. After centrifugal separation of the crude lysate, the GST-UBLCP1 was present predominantly in the soluble fraction. The fusion proteins were purified from the sonication supernatant of bacterial lysate by glutathione–Sepharose 4B affinity chromatography and analyzed by 10% SDS–PAGE. One major protein band corresponding to the expected 62 kDa recombinant protein was detected in elution fractions (Fig. 4).
Fig. 5. Enzymatic properties of UBLCP1. (A) Reaction mixtures containing 50 mM Tris acetate (pH 5.0), 20 mM pNPP, 10 mM MgCl2, and GSTUBLCP1 proteins as specified were incubated for 1 h at 37 C. (B) Effect of pH on the activity of UBLCP1. Reaction mixtures containing 50 mM Tris acetate (pH 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5), 10 mM MgCl2, 20 mM pNPP, and 10 lg UBLCP1 were incubated for 1 h at 37 C. (C) Effect of divalent cations on UBLCP1 activity. Reaction mixtures containing 50 mM Tris acetate (pH 5.0), 20 mM pNPP, 10 lg UBLCP1, and 10 mM each of the divalent cations.
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UBLCP1 is a CTD phosphatase with possible preference for Ser-5 GST-UBLCP1 fusion protein showed phosphatase activity toward p-nitrophenyl phosphate. The p-nitrophenyl reaction product was detected via its absorbance at 420 nm, which was directly proportional to the concentration of the recombinant protein (Fig. 5A). As reported for FCP1 from Schizosaccharomyces pombe and SCP1 utilizing pNPP as substrate [10,15], the optimal pH for UBLCP1 enzymatic activity is also near 5.0 (Fig. 5B). When using synthetic CTD peptides as substrate, the optimal pH for S. pombe FCP1 was also the same [10]. This unusual property might be ascribed to the catalytic mechanism of DXDX (T/V) family of phosphotransferases, which calls for an unprotonated aspartate nucleophile and a protonated aspartate general acid catalyst [20,24]. The phosphatase activity of UBLCP1 was Mg2+ dependent and Ca2+ could not substitute for Mg2+. Mn2+ also showed effect on increasing the activity, whereas Cu2+ and Zn2+ showed little effects on the activity (Fig. 5C). To determine whether UBLCP1 has CTD phosphatase activity and whether it displays any preference for Ser-2 or Ser-5, we assayed purified GST-UBLCP1 using a GST-CTD substrate, which had been phosphorylated using Cdc2 protein kinase. The GST-CTDo was detected by Western blot using H5 or H14 antibody. The results showed that UBLCP1 could effectively dephosphorylate Ser-5 site within CTD repeat. However, it seems UBLCP1 has relatively low activity on Ser-2 site (Fig. 6). Thus, these results indicated that UBLCP1 is a CTD phosphatase with possible preference for Ser-5 in vitro. Although its CTD phosphatase activity against RNAPII has not been validated in vivo, it is reasonable to speculate that UBLCP1 may play a role in regulating the phosphorylation state of RNAP II. During the transcription cycle, Ser-5 phosphorylation of RNAP II CTD is mainly detected at the promoter region, whereas Ser-2 phosphorylation is present in coding regions [25]. Given the possible preference of UBLCP1 for Ser-5, UBLCP1 is a candidate for acting early in the transcription cycle.
Fig. 6. UBLCP1 dephosphorylates GST-CTD. The GST-CTD was phosphorylated at both sites of Ser-2 and Ser-5 by Cdc2 kinase. The indicated amounts of GST-UBLCP1 or GST alone were added to each reaction. Phosphorylation status of CTD was detected by immunoblotting with H14 and H5, which is specific to Ser-5-PO4 and Ser-2-PO4, respectively.
Different from those CTD phosphatases previously identified, UBLCP1 has a ubiquitin-like domain in N-terminal, which contains a proteasome-interacting motif, indicating its potential ability of interacting with proteasome. In fact, our unpublished data showed that UBLCP1 could interact with a subunit of proteasome in vitro. Recent studies have revealed a number of connections between RNA polymerase II transcription and the proteasome [26,27]. Especially, it has been shown that the 26S proteasome and RNAP II can interact physically and functionally in vivo [28]. For the present, we cannot surmise the function of UBLCP1 with confidence, but considering the combination of the potential abilities of UBLCP1 to regulate the phosphorylation status of RNA polymerase II and to interact with the proteasome, we cautiously speculate that UBLCP1 may function in a certain period of transcriptional process, in which the proteasome might be involved.
Acknowledgments We thank Dr. William Dynan for the kind gift of the GST-CTD plasmid. We also thank Dr. Michael E. Dahmus for beneficial communication. This work was supported by a grant (10490193) from the Major Programs of the National Natural Science Foundation of China.
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