Although RecQ5β is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5β is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5β was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG–RNAP II 33 kDa subunit or FLAG–Pin1. Different regions of the non-helicase domain of the RecQ5β molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5β was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and β-actin genes. Knockdown of the RecQ5β transcript increased the transcription of those genes. The results of the present study suggest that RecQ5β has suppressive roles in events associated with RNAP II-dependent transcription.

INTRODUCTION

The proteins belonging to the RecQ family are distributed among a wide range of species, from unicellular organisms such as Escherichia coli and Saccharomyces cerevisiae to humans. They all have a ∼450 amino acid core domain containing seven helicase motifs, including a DEXH box in motif II, and many of them have sequence extensions at their N- and/or C-termini [1]. The human RecQ helicase family has five members: RecQ1 (RecQL), BLM (Bloom syndrome helicase), WRN (Werner syndrome helicase), RecQ4 and RecQ5. Defects in three of these genes, WRN, BLM and RecQ4, causes Werner syndrome, Bloom syndrome and Rothmund–Thomson syndrome respectively [25]. RecQ family members seem to be functionally conserved throughout evolution with regards to various aspects of DNA editing pathways [69]. The disease-associated RecQ helicases interact directly with the nuclear proteins required for chromosome maintenance, including the homologous recombination mediator protein RAD52, the cell-cycle regulator p53, the breast cancer-associated protein BRCA1 (breast-cancer susceptibility gene 1) and the ssDNA (single-stranded DNA)-binding replication protein A [1014]. Although common functional similarities have been reported among the human RecQ homologues [4], functional variations have also been identified; for instance, BLM suppresses cell growth in the yeast top3 sgs1 mutant strain and restores increased sensitivity of the sgs1 mutant to hydroxyurea, but WRN does not [15]. WRN is thought to play a role in RNAP II (RNA polymerase II)-dependent transcription, possibly as a transcriptional activator, although a physical interaction between RNAP II and WRN has not been demonstrated [16]. In contrast, the involvement of BLM in transcription has not yet been reported. These similarities and differences probably reflect the overlapping and distinctive clinical phenotypes between Bloom and Werner syndromes.

No human genetic disorder has been associated with the two RecQ genes RECQ1 and RECQ5. The RecQ5 helicase has a low molecular mass, similar to the RecQ1 helicase, and was shown to be expressed in all organs examined [17]. RecQ5β has the largest polypeptide of the three RecQ5 isoforms (α, β and γ), which are generated by alternative splicing [18]. RecQ5β is localized to the nucleoplasm, which is in contrast with the α and γ isoforms, both of which are principally localized to the cytoplasm. RecQ5β is a ssDNA-stimulated ATPase and an ATP-dependent DNA helicase and interacts with topoisomerase 3 [1921]. Although RecQ5β is implicated in maintenance of DNA stability and repair [22,23], the cellular function of RecQ5β remains to be explored. In the present study, we show that RecQ5β is a component of the RNAP II-associated complex and is associated with the coding regions of the LDL (low-density lipoprotein) receptor and β-actin genes.

EXPERIMENTAL

Materials

The HEK-293 (human embryonic kidney-293) EBNA cell line (expressing the Epstein–Barr virus nuclear antigen), OptiMEM I and Lipofectamine™ 2000 were obtained from Invitrogen. DMEM (Dulbecco's modified Eagle's medium), anti-FLAG M2 antibody–agarose, mouse monoclonal anti-FLAG M2 antibody, rabbit polyclonal anti-FLAG antibody, FLAG peptide, Igepal CA-630, RNase A, α-cyano-4-hydroxycinnnanic acid, mouse monoclonal anti-tubulin β, mouse monoclonal IgG1 and rabbit polyclonal IgG antibodies were obtained from Sigma–Aldrich. Vectashield mounting medium was purchased from Vector Laboratories. PVDF membranes, protein G–Sepharose 4 Fast Flow, horseradish-peroxidase-conjugated sheep anti-mouse and horseradish-peroxidase-conjugated donkey anti-rabbit IgG antibodies were obtained from Amersham Biosciences. Alkaline phosphatase-conjugated anti-mouse IgG and anti-rabbit antibodies were obtained from Cell Signaling Technology, and alkaline phosphatase-conjugated anti-goat IgG antibody was obtained from ICN Pharmaceuticals. The antibody against hypophosphorylated RNAP II (8WG16) was obtained from COVANCE. The antibody against RNAP IIO (hyperphosphorylated RNAP II large subunit) (MARA3) was a gift from Dr Bart Sefton (Molecular and Cell Biology Laboratory, The Salk Institute, La Jolla, CA, U.S.A.). The rabbit polyclonal anti-RNAP II (N-20), goat polyclonal anti-PABP1 (poly-A binding protein 1), goat polyclonal anti-U2AF65 (N-14), goat polyclonal anti-hnRNP [heterogeneous nuclear RNP (ribonucleoprotein)] A1 and goat polyclonal anti-lamin B antibodies were obtained from Santa Cruz Biotechnology. The mouse monoclonal antihnRNP C1/C2 antibody was obtained from Immunoquest (York, MD, U.S.A.). Alexa Fluor® 488-conjugated rabbit antimouse IgG antibody was obtained from Molecular Probes. Trypsin (sequencing grade) was obtained from Promega and Achromobacter lyticus protease l was obtained from WAKO Pure Chemicals (Osaka, Japan). ZipTipC18 was obtained from Millipore. DNase I was obtained from TaKaRa. KOD-plus DNA polymerase was obtained from TOYOBO. All other reagents were purchased from WAKO Pure Chemicals.

Cell culture

HEK-293 EBNA cells and HeLa cells were grown in DMEM supplemented with 100 i.u./ml penicillin G, 100 μg/ml streptomycin and 10% (v/v) heat-inactivated fetal calf serum. All cell cultures were maintained at 37 °C and 5% CO2. Actinomycin D was added to the medium at a final concentration of 5 μg/ml and incubated for 3 h where indicated in the Figure legends.

Preparation of anti-RecQ5β antibody-fixed protein G–Sepharose bead columns

The mouse monoclonal antibody against human RecQ5β was prepared by immunization of mice with a purified recombinant C-terminal fragment of RecQ5β (amino acid residues 848–991) [24]. Antibody-fixed protein G–Sepharose was prepared as described previously [25]. Briefly, RecQ5β monoclonal antibody (40 μg) was mixed with 40 μl of a slurry of protein G–Sepharose 4 Fast Flow in 1 ml of PBS and incubated at room temperature (20 °C) for 1 h with gentle mixing. The beads were washed three times with 1 ml of 0.15 M sodium borate buffer (pH 9.0), centrifuged at 1000 g for 5 min and resuspended in 1 ml of 0.15 M sodium borate buffer. The protein G-bound anti-RecQ5β antibody was bound covalently by incubating with 20 mM dimethyl pimelimidate (Pierce) for 30 min at room temperature with gentle mixing. The reaction was stopped by the addition of 0.2 M ethanolamine (pH 8.0). The protein G–Sepharose beads fixed with anti-RecQ5β antibody were washed with PBS and stored in PBS containing 0.05% sodium azide until use. Protein G–Sepharose beads fixed with unspecified IgG1 antibody was prepared following the same method.

Preparation of the RecQ5β-associated complexes

Confluent human HEK-293 EBNA cells were washed with PBS and suspended in five packed cell volumes of lysis buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2 and 0.5% Igepal CA-630] containing 0.25 M sucrose, 1 mM NaF, 1 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml pepstatin A and 2 μg/ml leupeptin for 10 min on ice and lysed by sonication (output 20, 6×4 s, Model 250 sonifier; Branson Sonic Power Company, Danbury, CT, U.S.A.). After removal of any insoluble residue by centrifugation at 20000 g for 30 min at 4 °C, the soluble cell lysates (5000 μg) obtained from 107 cells were pre-treated first with Sepharose CL-6B beads and then with protein G–Sepharose 4 Fast Flow to remove proteins that bound non-specifically to Sepharose beads or protein G before immunoprecipitation of RecQ5β. The pre-cleared cell lysates were then incubated with 40 μl of anti-RecQ5β antibody-fixed protein G–Sepharose beads for >4 h at 4 °C. After incubation, the RecQ5β complexes bound to the beads were washed five times with 1 ml of lysis buffer and washed twice with 1 ml of buffer containing 50 mM Tris/HCl (pH 8.0), 150 mM NaCl and 5 mM MgCl2. The RecQ5β-associated proteins were eluted by incubation with 40 μl of 0.1 M glycine buffer (pH 1.5) for 30 min on ice, and added to 2% (w/v) SDS sample buffer and subjected to SDS/PAGE.

Protein identification by PMF (peptide mass fingerprinting)

SDS/PAGE gel bands containing proteins were excised and subjected to in-gel protease (trypsin) digestion as described by Yanagida et al. [26]. Peptides generated by digestion were recovered, purified with ZipTipC18 and analysed by the PMF method using a MALDI-TOF–MS (matrix-assisted laser-desorption ionization–time-of-flight MS) (Voyager DE-STR, Applied Biosystems) [27]. Peptide masses were searched using the database fitting program MS-Fit with 50 p.p.m. mass accuracy (http://prospector.ucsf.edu), and protein identification was performed using the criteria described previously [28].

Immunoblot analysis

Proteins obtained by immunopurification were separated by SDS/PAGE, transferred on to PVDF membranes and treated with TBST [TBS (Tris-buffered saline) containing 0.1% Tween 20] containing 5% (w/v) non-fat dried skimmed milk powder. The membrane was then incubated for 2 h at room temperature with the primary antibody [anti-RNAP II antibodies (N-20, 1:200 dilution; 8WG16, 1:700 dilution; MARA3, 1:10 dilution), anti-RecQ5β antibody (5 μg/ml), anti-FLAG antibody (1:5000 dilution), anti-PABP1 antibody (1:200 dilution), anti hnRNP A1 antibody (1:200 dilution), anti-lamin B antibody (1:200 dilution), anti-hnRNP C1/C2 antibody (1:1000 dilution), anti-U2AF65 antibody (1:200 dilution) and anti-tubulin β antibody (1:10000 dilution)], washed three times with TBST, and then incubated for 1 h at room temperature with alkaline phosphatase-conjugated secondary antibodies [anti-mouse IgG; anti-rabbit IgG and anti-goat IgG (all at 1:5000 dilution)] or horseradish peroxidase-conjugated secondary antibodies [sheep anti-mouse and donkey anti-rabbit IgG antibodies (both at 1:5000 dilution)]. After incubation, the membrane was washed three times with TBST and once with TBS. The bound secondary antibody was visualized with NBT (Nitro Blue Tetrazolium)/BCIP (5-bromo-4-chloroindol-3-yl phosphate) solution (Roche) or by ECL® (enhanced chemiluminescence) Plus Western blotting detection system (GE Healthcare).

RNAi (RNA interference)

For silencing of the RecQ5β transcript, we used Stealth Select siRNA (small interfering RNA) oligonucleotides (Invitrogen), directed to nucleotides 1902–1926 of the RecQ5β mRNA 5′-UGGAAUGUCAUACUCAUUGGGCUCC-3′ and 5′-GGAGCCCAAUGAGUAUGACAUUCCA-3′. The scrambled Stealth Select RNAi oligonucleotides [scRNA (scrambled control RNA); 5′-UGGGCAAUGUUACUCAUACGGUUCC-3′ and 5′-GGAACCGUAUGAGUAACAUUGCCCA-3′] were used as a negative control. The siRNA or scRNA were transfected at a concentration of 500 nM using Lipofectamine™ 2000 into HEK-293 EBNA cells that had been cultured in 35 mm tissue-culture dishes. After incubation for 5 days, the siRNA- or scRNA-transfected cells were harvested and subjected to immunoprecipitation.

Immunodepletion assay

The RecQ5β complexes were immunodepleted from HEK-293 EBNA cell lysates by incubating with 20 μl of anti-RecQ5β antibody-fixed protein G–Sepharose beads, and then the RecQ5β-immunodepleted cell lysates were re-immunoprecipitated using anti-RecQ5β antibody-fixed protein G–Sepharose beads. As a control, HEK-293 EBNA cell lysates immunodepleted using unspecified IgG1-fixed protein G–Sepharose were re-immunoprecipitated with anti-RecQ5β antibody-fixed protein G–Sepharose beads.

DNase or RNase treatment

The RecQ5β-associated complexes immunoprecipitated with the anti-RecQ5β antibody-fixed protein G–Sepharose beads or with anti-FLAG M2 antibody–agarose were washed five times with lysis buffer and treated with 160 units/ml DNase I in 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl and 8 mM MgCl2, or treated with 100 units/ml RNase A in 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl at 37 °C for 30 min. Complexes were then washed three times with lysis buffer, twice with 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl, and eluted using 2% (w/v) SDS sample buffer.

Immunoprecipitation of RNAP II-associated complexes

HEK-293 EBNA cell lysates (2 μg/μl) obtained as described above were immunoprecipitated with anti-RNAP II antibodies (N-20, 3 μg; 8WG16, 3 μg; MARA3, 1:10 dilution) overnight at 4 °C. The antibody-bound RNAP II-associated complexes were incubated with protein A–Sepharose beads (20 μl) for 2 h at 4 °C, and then the complexes–beads were washed five times with lysis buffer, twice with 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl, and eluted using 2% (w/v) SDS sample buffer.

Construction of epitope-tagged expression plasmids

For the construction of FLAG-tagged RPB3 (RNAP II 33 kDa subunit) (FLAG–RPB3) and FLAG-tagged Pin1 (FLAG–Pin1), cDNAs derived from the HeLa cell line and the pGEX-4T-1-Pin1 plasmid [29] were used as templates respectively. cDNAs for FLAG–RPB3 and FLAG–Pin1 were obtained by PCR with the following primer sets: 5′-TATATAAAGCTTGCCACCATGGACTACAAGGACGACGACGACAAGATGCCGTACGCCAACCAGCCTACC-3′ and 5′-TATATAGGATCCTTAATTTATGGTTAGCACATCACT-3′ for RPB3; and 5′-ATTGAGCTAGCGCCACCATGGACTACAAGGACGACGACGACAAGATGGCGGACGAGGAGAAACTG-3′ and 5′-TCCGAATTCTCACTCAGTGCGGAGGATGAT-3′ for Pin1. The PCR products were digested with HindIII and BamHI for FLAG–RPB3 and with EcoRI and NheI for FLAG–Pin1, and cloned into the HindIII and BamHI or EcoRI and NheI sites of the pcDNA3.1 expression vector to generate FLAG–RPB3 and FLAG–Pin1.

Human RecQ5β cDNA was isolated from a human testis cDNA library and cloned into pAP3-neo [19]. In the pBluescriptII KS+ background, the sequence immediately upstream of the translational start site (the first ATG codon) of RecQ5β was modified to create a NheI restriction site, such that the intact ORF (open reading frame) could be excised using NheI and XhoI. The NheI- and XhoI-digested DNA fragment derived from the RecQ5β cDNA was sub-cloned into the NheI and XhoI site of the pcDNA3 mammalian expression plasmid containing a FLAG epitope sequence.

The expression plasmids for RecQ5β β533 and β917 (see Figure 5A) truncation mutants were constructed by PCR using the following primer sets: 5′-TATATAAAGCTTGCCACCATGGCCGACTACAAGGAC-3′ and 5′-TATATACTCGAGTCAGCTGGTTACCTTTCTCTTCTTCTTAGGGAATTCGGGACAGTTCTCATCTGGGGGTAC-3′ for β533; and 5′-TATATAAAGCTTGCCACCATGGCCGACTACAAGGAC-3′ and 5′-TATATACTCGAGTCAATTTGCAGCCT CCTTCAAGGAGAC-3′ for β917. FLAG–RecQ5β in pcDNA3 was used as the template. The amplified DNA fragments were digested with HindIII and XhoI and sub-cloned into pcDNA3.1. β533 in pcDNA3.1 was digested with HindIII and EcoRI and used to construct other deletion mutants, such as NLS-pcDNA3.1 (where NLS is nuclear localization signal). The expression plasmids for other truncation mutants [β395, β617, β744, βc2, βc3, βc4 and βc34 (see Figure 5A)] were constructed by PCR using primer sets as follows: 5′-TATATAAAGCTTGCCACCATGGCCGACTACAAGGAC-3′ and 5′-TATATAGAATTCAGTGGCTTTATCAGATGCTTTGTT-3′ for β395; 5′-TATATAAAGCTTGCCACCATGGCCGACTACAAGGAC-3′ and 5′-TATATAGAATTCGGGCTGCCCATCCTTGGAGGCTCT-3′ for β617; 5′-TATATAAAGCTTGCCACCATGGCCGACTACAAGGAC-3′ and 5′-TATATAGAATTCGCCCTTGGCAAGGGAGCTGCCCCC-3′ for β744; 5′-TATATAAAGCTTGCCACCATGGACTACAAGGACGACGACGACAAGTATGACATGGGAGGCAGTGCCAAG-3′ and 5′-TATATAGAATTCGCCCTTGGCAAGGGAGCTGCCCCC-3′ for βc2; 5′-TATATAAAGCTTGCCACCATGGACTACAAGGACGACGACGACAAGCTGAAAGAGGCTTCTAGCAGGAGG-3′ and 5′-TATATAGAATTCGGGCTGCCCATCCTTGGAGGCTCT-3′ for βc3; 5′-TATTAAAGCTTGCCACCATGGACTACAAGGACGACGACGACAAGATCATGGCCTTTGATGCCCTGGTG-3′ and 5′-TATATAGAATTCGGGACAGTTCTCATCTGGGGGTAC-3′ for βc4; and 5′-TATTAAAGCTTGCCACCATGGACTACAAGGACGACGACGACAAGATCATGGCCTTTGATGCCCTGGTG-3′ and 5′-TATATAGAATTCGGGCTGCCCATCCTTGGAGGCTCT-3′ for βc34. FLAG–RecQ5β in pcDNA3 was used as the template. The amplified DNA fragments were digested with HindIII and EcoRI and sub-cloned into NLS-pcDNA3.1. To construct the βc1 expression plasmid, FLAG–RecQ5β DNA in pcDNA3 was digested with NheI to remove the 5′ region upstream of the endogenous NheI site in RecQ5β DNA and was self-ligated to produce a deletion mutant that encodes the C-terminal 246-amino- acid polypeptide of RecQ5β. For localization of β395, β533, β617, β744, βc2, βc3, βc4 and βc34 to the nucleus, the NLS (PKKKRKV) encoded by the SV40 (simian virus 40) large T-antigen was inserted at the C-terminus. The expression plasmid for βdHE was constructed by PCR using the primer set as follows: 5′-TATATAAGCTTACCATGGATTACAAGGATGACGACGATAAGGAATTCATCATGGCCTTTGATGCCCTG-3′ and 5′-ATATACTCGAGTCATCTCTGGGGGCCACACAG-3′. The amplified DNA fragments were digested with HindIII and XhoI and sub-cloned into pcDNA3.1.

Immunoprecipitation of FLAG-tagged protein complexes

HEK-293 EBNA cells were transfected with the indicated expression plasmids using either the calcium phosphate method or Lipofectamine™ 2000 and grown for 24 h at 37 °C [2628]. The transfected cells obtained were washed with PBS, lysed with lysis buffer and sonicated as described above. The cell lysates were obtained by centrifugation at 20000 g for 30 min at 4 °C. For immunoprecipitation, the cell lysates were incubated with 20 μl of anti-FLAG M2 antibody–agarose for 4 h at 4 °C. The protein-bound M2–agarose beads were washed five times with lysis buffer and washed twice with 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl, and the immunoprecipitates were eluted using 50 mM Tris/HCl (pH 8.0) containing 150 mM NaCl and 500 μg/ml FLAG peptide.

Chromatin and nuclear matrix preparation

Chromatin and nuclear matrix fractions were prepared according to a method described previously [30]. Briefly, HEK-293 EBNA cells were washed with PBS, lysed in CSK buffer [cytoskeletal buffer; 10 mM Pipes (pH 6.8), 100 mM NaCl, 300 mM sucrose and 3 mM MgCl2] supplemented with 0.5% Triton X-100, 1 mM EGTA, 1 mM DTT (dithiothreitol), 1 mM PMSF, 2 μg/ml pepstatin A and 2 μg/ml leupeptin for 10 min on ice and centrifuged at 7500 g for 3 min. The supernatant was used as the Triton X-100-soluble fraction. The pellet was resuspended in DNase digestion buffer [10 mM Pipes (pH 6.8), 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100 and 1 mM PMSF] in the presence of 800 units/ml DNase I and 20 units of RNase inhibitor (WAKO Pure Chemicals) and incubated for 1 h at 37 °C. After addition of ammonium sulfate to a final concentration of 250 mM, the DNase I-treated pellet suspension was incubated for 10 min on ice and centrifuged at 7500 g for 5 min. The supernatant was used as the chromatin fraction. The pellet was extracted using urea buffer [50 mM Tris/HCl (pH 7.0), 9 M urea and 1.05% (v/v) 2-mercaptoethanol], followed by centrifugation at 20000 g for 10 min. The supernatant was used as the nuclear matrix fraction. HEK-293 EBNA cell lysates were extracted with urea buffer and centrifuged at 20000 g for 10 min, and the supernatant was used as the whole cell lysate.

Ultracentrifugation of RecQ5β-associated complexes

HEK-293 EBNA cell lysates were fractionated to obtain the RNP-containing fraction [31]. Briefly, cells collected from dishes were washed with PBS, resuspended in five packed column volumes of RSB-100 buffer [10 mM Tris/HCl (pH 7.4), 100 mM NaCl and 3 mM MgCl2] containing 0.5% Triton X-100, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM PMSF, 10 mM NaF and 1 mM sodium vanadate, and then sonicated twice for 5 s each (Model 250 sonifier). The sonicated materials were layered on to a 30% (v/v) sucrose cushion in RSB-100 buffer and centrifuged at 4000 g for 15 min. The supernatant was collected and used as the RNP-containing fraction. The RNP-containing fraction obtained was overlaid on a 10–40% (v/v) sucrose gradient formed using an ISCO gradient former (Model 160; Teledyne Isco, Lincoln, NE, U.S.A.), separated by ultracentrifugation at 45000 rev./min for 219 min at 4 °C using an MLS50 rotor (Beckman Coulter) and ten fractions (500 μl) were collected. Ribosomal subunits (40 S/60 S/80 S) were used to estimate the size of the RecQ5β-associated complexes. Consecutive pairs of fractions collected from the sucrose gradient were pooled, and the RecQ5β-associated complexes were immunoprecipitated with anti-RecQ5β antibody-fixed protein G–Sepharose beads. Proteins in each pooled fraction were also precipitated using 10% (v/v) TCA (trichloroacetic acid), adjusted to a pH of approx. 7, dissolved in 2% (w/v) SDS sample buffer and subjected to Western blot analysis.

Immunocytochemistry

Cells were cultured on eight-well slides (Biocoat; Becton Dickinson Labware) and transfected with the indicated expression plasmid using Lipofectamine™ 2000. The transfected cells or intact cells were fixed with 3.7% (w/v) formaldehyde in PBS for 10 min. After washing with PBST (PBS containing 0.05% Tween 20), the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, and treated with 3% (w/v) non-fat dried skimmed milk powder in PBS for 1 h. The cells were incubated with the indicated primary antibody [mouse anti-FLAG antibody or anti-RecQ5β antibody (10 μg/ml for both)] for 2 h. After washing three times with PBST for 10 min, the cells were further incubated with the indicated secondary antibody [Alexa Fluor® 488-conjugated anti-mouse IgG antibody (10 μg/ml)] for 1h. After incubation, cells were washed three times with PBST for 10 min and mounted with Vectashield containing DAPI (4′,6-diamidino-2-phenylindole). Fluorescence images were visualized with an Axiovert 200 M microscope (Carl Zeiss, Germany).

ChIP (chromatin immunoprecipitation) assay

After 24 h of transfection of FLAG-RecQ5β expression plasmid into HEK-293 EBNA cells, 5×106 cells were cross-linked with 1% formaldehyde at room temperature for 10 min. The excess formaldehyde was quenched with 0.125 M glycine for 10 min. The cross-linked cells were washed twice with PBS and incubated in 100 μl of 50 mM Tris/HCl (pH 8.0) buffer containing 1% SDS, 10 mM EDTA, 1 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin A and 1 mM NaF for 10 min on ice. The cells were sonicated further (10×20 s, 20% amplitude; Branson sonifier W-250 D), which was expected to produce ∼500 nt chromatin fragments, and were centrifuged at 20000 g for 10 min. The supernatant was diluted 1:10 using ChIP dilution buffer [16.7 mM Tris/HCl (pH 8.0) containing 0.01% SDS, 1.1% (v/v) Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 1 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin A and 1 mM NaF]. An aliquot (30 μl) of the diluted solution was retained to be used as a control. The remaining solution was pre-cleared with protein A–Sepharose beads (saturated with 0.2 mg/ml salmon sperm DNA and 0.5 mg/ml BSA) at 4 °C for 1 h, and incubated with either 3 μg of the RNAP II antibody N-20, 3 μg of rabbit polyclonal anti-FLAG antibody or 3 μg of unspecified rabbit IgG antibody (negative control) at 4 °C overnight. The antibody was pulled down using protein A–Sepharose beads by incubating at 4 °C for 1 h. The antibody-bound beads were washed by incubating for 3 min with each of the following buffers: (i) 20 mM Tris/HCl (pH 8.1) containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA and 150 mM NaCl (low-salt immune complex wash buffer); (ii) 20 mM Tris/HCl (pH 8.1) containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA and 500 mM NaCl (high-salt immune complex wash buffer); (iii) 10 mM Tris/HCl (pH 8.1) containing 0.1% SDS, 1% Triton X-100, 1 mM EDTA, 0.25 M LiCl, 1% Igepal CA-630 and 1% deoxycholic acid (lithium chloride immune complex wash buffer) and (iv) 10 mM Tris/HCl and 1 mM EDTA (TE buffer) twice. The antibody-bound chromatin was eluted with 0.1 M NaHCO3 containing 1% SDS and 10 mM DTT, the formaldehyde cross-linking was reversed by heating at 65 °C for 6 h, and digested with 0.2 unit of proteinase K (Invitrogen) for 1 h at 45 °C. The released DNA was purified using the Qiagen PCR purification kit following the manufacturer's instructions and used for amplification of target sequences by PCR with KOD-plus DNA polymerase using primer sets; 5′-TGTTAACAGTTAAACATCGAGAA-3′ and 5′-CCCGCGATTGCACTCGGGGC-3′ for LDL receptor promoter; 5′-ATCTCCTCAGTGGCCGCCTCTACTG-3′ and 5′-CAGTTTTCTGCGTTCATCTTGGCTTGA-3′ for LDL receptor coding region; 5′-GAGCACAGAGCCTCGCCTTT-3′ and 5′-AGACAAAGACCCCGCCGGTT-3′ for β-actin promoter; 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′ for β-actin coding region; and 5′-TTGAGAAGCCTTCGCTTCGAAG-3′ and 5′-TTGCGCTGCATGTGCCATTAAG-3′ for mitochondrial DNA. The intensity of band staining on an agarose gel after electrophoresis was quantified using the ImageJ program (National Institutes of Health).

Quantitative real-time PCR

The siRNA or scRNA oligonucleotides (40 nM) described above were transfected into HeLa cells using Lipofectamine™ 2000 and cultured in 24-well plates. After incubation for 96 h, total cellular RNA was extracted from the siRNA- or scRNA-transfected cells by using the Total RNA Isolation System (Promega) according to the manufacturer's instructions. The knockdown of RecQ5β was confirmed by immunoblot analysis. ssDNA was synthesized with a random hexamer primer using the PrimeScript RT reagent kit (TaKaRa) according to the manufacturer's instructions. Quantitative real-time PCR was carried out using the Thermal Cycler Dice Real Time System (TaKaRa) with the primer sets: 5′-AGTTGGCTGCGTTAATGTGAC-3′ and 5′-TGATGGGTTCATCTGACCAGT-3′ for LDL receptor; 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′ for β-actin; and 5′- ATTGATCGCCAGGGTTGATT-3′ and 5′- CGGGGATGGTCGTCCTCTTC-3′ for 7SK RNA. The relative amount of RNA was estimated using standard curve methods and normalized to 7SK RNA.

RESULTS AND DISCUSSION

Isolation of RecQ5β-associated complexes and MS-based identification of their components

We used a mouse monoclonal antibody against the C-terminal fragment of RecQ5β (amino acid residues 848–991) [24] to isolate protein complexes associated with endogenously expressed RecQ5β in HEK-293 EBNA cells. We first carried out identification of the protein components of the immunoprecipitated RecQ5β-associated complex by excision of individual bands from SDS/PAGE gels, followed by PMF using MALDI-TOF–MS. We identified at least four subunits of RNAP II in the isolated RecQ5β-associated complex (Figure 1 and Supplementary Table 1 at http://www.BiochemJ.org/bj/413/bj4130505add.htm). To confirm the specificity of the antibody against RecQ5β, incubation with siRNA against RecQ5β and immunoprecipitation was performed. The interaction with the RNAP II large subunit was reduced in the presence of the decreased amount of RecQ5β remaining after RNAi (Figure 1B), indicating that RecQ5β interacts specifically with the RNAP II large subunit. In addition, the RNAP II large subunit was no longer co-immunoprecipitated with the anti-RecQ5β antibody after depletion of RecQ5β with an anti-RecQ5β antibody, despite most of the RNAP II large subunit remaining in the cell extract (Figure 1C); thus the antibody alone has no association with the RNAP II subunit.

Isolation of RecQ5β-associated complexes and identification of RecQ5β-binding proteins

Figure 1
Isolation of RecQ5β-associated complexes and identification of RecQ5β-binding proteins

(A) RecQ5β-associated complexes were isolated by immunoprecipitation from HEK-293 EBNA cell lysates (5000 μg) with anti-RecQ5β antibody-fixed beads. The immuno-isolated complexes (RecQ5β) were resolved by SDS/PAGE (10% gels) followed by silver staining. The immunoprecipitates obtained with unspecified IgG1-fixed beads were used as a negative control (IgG). Cell lysates (20 μg) were also resolved as a control (Input). Molecular-mass markers are indicated at the left-hand side (in kDa). Each protein band was excised, subjected to in-gel protease digestion and analysed by MALDI-TOF–MS. The resulting PMFs were matched to a database and the identified proteins are indicated on the right-hand side. (B) RecQ5β-associated complexes were immunoprecipitated (IP) from siRNA (si) or scRNA (sc)-transfected (TF) HEK-293 EBNA cells using the anti-RecQ5β antibody, and then RecQ5β and RNAP II were detected by immunoblot (IB) analysis using the anti-RecQ5β antibody and the anti-RNAP II antibody (N-20). As a control, the immunoprecipitates obtained by incubation with unspecified IgG1 antibody were also analysed by immunoblotting. For immunoprecipitation, cell lysates (1000 μg) were used and pre-immunoprecipitation cell lysates (50 μg) were also analysed by immunoblotting as a control (Input). (C) The immunoprecipitated (IP) complexes were removed from HEK-293 EBNA cell lysates by immunoprecipitation with anti-RecQ5β antibody (+) or unspecified IgG antibody (−) to immunodeplete (ID) the cell lysates. These immunodepleted cell lysates (300 μg) were subsequently immunoprecipitated using the anti-RecQ5β antibody, and the immunoprecipitates obtained were analysed by immunoblotting (IB) using the anti-RecQ5β and N-20 antibodies. As a control, immunoprecipitation with unspecified IgG1 antibody was also performed. Immunodepleted cell lysates (30 μg) were also analysed as a control (Input).

Figure 1
Isolation of RecQ5β-associated complexes and identification of RecQ5β-binding proteins

(A) RecQ5β-associated complexes were isolated by immunoprecipitation from HEK-293 EBNA cell lysates (5000 μg) with anti-RecQ5β antibody-fixed beads. The immuno-isolated complexes (RecQ5β) were resolved by SDS/PAGE (10% gels) followed by silver staining. The immunoprecipitates obtained with unspecified IgG1-fixed beads were used as a negative control (IgG). Cell lysates (20 μg) were also resolved as a control (Input). Molecular-mass markers are indicated at the left-hand side (in kDa). Each protein band was excised, subjected to in-gel protease digestion and analysed by MALDI-TOF–MS. The resulting PMFs were matched to a database and the identified proteins are indicated on the right-hand side. (B) RecQ5β-associated complexes were immunoprecipitated (IP) from siRNA (si) or scRNA (sc)-transfected (TF) HEK-293 EBNA cells using the anti-RecQ5β antibody, and then RecQ5β and RNAP II were detected by immunoblot (IB) analysis using the anti-RecQ5β antibody and the anti-RNAP II antibody (N-20). As a control, the immunoprecipitates obtained by incubation with unspecified IgG1 antibody were also analysed by immunoblotting. For immunoprecipitation, cell lysates (1000 μg) were used and pre-immunoprecipitation cell lysates (50 μg) were also analysed by immunoblotting as a control (Input). (C) The immunoprecipitated (IP) complexes were removed from HEK-293 EBNA cell lysates by immunoprecipitation with anti-RecQ5β antibody (+) or unspecified IgG antibody (−) to immunodeplete (ID) the cell lysates. These immunodepleted cell lysates (300 μg) were subsequently immunoprecipitated using the anti-RecQ5β antibody, and the immunoprecipitates obtained were analysed by immunoblotting (IB) using the anti-RecQ5β and N-20 antibodies. As a control, immunoprecipitation with unspecified IgG1 antibody was also performed. Immunodepleted cell lysates (30 μg) were also analysed as a control (Input).

We also examined the presence of RNAP II in the RecQ5β-associated complex by immunoblot analysis with three available antibodies against the RNAP II large subunit (N-20, 8WG16 and MARA3) [32,33] and showed that RecQ5β was associated with both RNAP IIA (hypophosphorylated RNAP II large subunit, which is recognized with 8WG16 and N-20 antibodies) and RNAP IIO (which is detected by all RNAP II antibodies used) (Figure 2A). Immunoblot analysis with the anti-RNAP II antibody N-20 showed that both RNAP IIA and RNAP IIO associated with RecQ5β after either DNase I or RNase A treatment (Figure 2B and Supplementary Figure 1 at http://www.BiochemJ.org/bj/413/bj4130505add.htm). These results indicate that neither DNA nor RNA is required to maintain an association between RecQ5β and the large subunit of RNAP II.

Immunoblot analysis of RNAP II subunits identified in the RecQ5β-associated complexes by MS-based analysis

Figure 2
Immunoblot analysis of RNAP II subunits identified in the RecQ5β-associated complexes by MS-based analysis

The three different RNAP II antibodies used recognize different epitopes: N-20 reacts with both RNAP IIA and RNAP IIO; 8WG16 mainly reacts with RNAP IIA and MARA3 reacts with RNAP IIO. (A) RecQ5β-associated complexes were isolated from HEK-293 EBNA cell lysates (2000 μg) by immunoprecipitation (IP) with the anti-RecQ5β antibody and were analysed by immunoblotting (IB) with the anti-RecQ5β and anti-RNAP II antibodies (N-20, 8WG16 and MARA3) indicated at the right-hand side. As a control, the lysates were also immunoprecipitated using unspecified IgG1-fixed beads (IgG) and was analysed by immunoblotting. Cell lysates (20 μg) were also immunoblotted as a control (Input). (B) RecQ5β-associated complexes which were isolated from HEK-293 EBNA cell lysates (1000 μg) by immunoprecipitation (IP) with anti-RecQ5β antibody were treated with (+) or without (−) either DNase I or RNase A, and then were analysed by immunoblotting (IB) with an anti-RecQ5β antibody or anti-RNAP II antibody (N-20). As a control, immunoprecipitation was also performed using unspecified IgG1 antibody and was analysed by immunoblotting. Cell lysates (50 μg) were also immunoblotted as a control (Input). (CE) RNAP II-associated complexes which were immunoprecipitated (IP) from HEK-293 EBNA cell lysates (1000 μg) using anti-RNAP II antibodies N-20 [3 μg; (C)], 8WG16 [3 μg; (D)] or MARA3 [1:10 dilution; (E)] were analysed by immunoblotting (IB) with an anti-RecQ5β antibody or anti-RNAP II antibody [N-20 (C), 8WG16 (D) and MARA3 (E)]. As a control, immunoprecpitation was also performed using mouse or rabbit IgG antibody (IgG) and was analysed by immunoblotting. Cell lysates (50 μg) were also immunoblotted as a control (Input). (F and G) HEK-293 EBNA cells were transfected (TF) with the mock pcDNA 3.1 plasmid (−) or FLAG-tagged expression plasmids expressing RPB3 (F) or Pin1 (G) (+). The immunopreciptates isolated from transfected HEK-293 EBNA cell lysates (2000 μg) by FLAG immunoprecipitation (IP) were analysed by immunoblotting (IB) with an anti-RNAP II antibody (N-20), anti-RecQ5β antibody or anti-FLAG antibody. Cell lysates (50 μg) were also immunoblotted as a control (Input). (H) HEK-293 EBNA cells (2×106 cells) were fractionated into three fractions (Triton soluble fraction, Chromatin fraction and Nuclear matrix fraction) and 3% of each fraction was subjected to Western blot analysis with anti-RNAP II (8WG16), anti-RecQ5β, anti-PABP1, anti-hnRNP A1 and anti-lamin B antibodies. The Triton soluble fraction, chromatin fraction and nuclear matrix fraction were indicated by the detection of PABP1, hnRNP A1 and lamin B respectively. IB, immunoblotting.

Figure 2
Immunoblot analysis of RNAP II subunits identified in the RecQ5β-associated complexes by MS-based analysis

The three different RNAP II antibodies used recognize different epitopes: N-20 reacts with both RNAP IIA and RNAP IIO; 8WG16 mainly reacts with RNAP IIA and MARA3 reacts with RNAP IIO. (A) RecQ5β-associated complexes were isolated from HEK-293 EBNA cell lysates (2000 μg) by immunoprecipitation (IP) with the anti-RecQ5β antibody and were analysed by immunoblotting (IB) with the anti-RecQ5β and anti-RNAP II antibodies (N-20, 8WG16 and MARA3) indicated at the right-hand side. As a control, the lysates were also immunoprecipitated using unspecified IgG1-fixed beads (IgG) and was analysed by immunoblotting. Cell lysates (20 μg) were also immunoblotted as a control (Input). (B) RecQ5β-associated complexes which were isolated from HEK-293 EBNA cell lysates (1000 μg) by immunoprecipitation (IP) with anti-RecQ5β antibody were treated with (+) or without (−) either DNase I or RNase A, and then were analysed by immunoblotting (IB) with an anti-RecQ5β antibody or anti-RNAP II antibody (N-20). As a control, immunoprecipitation was also performed using unspecified IgG1 antibody and was analysed by immunoblotting. Cell lysates (50 μg) were also immunoblotted as a control (Input). (CE) RNAP II-associated complexes which were immunoprecipitated (IP) from HEK-293 EBNA cell lysates (1000 μg) using anti-RNAP II antibodies N-20 [3 μg; (C)], 8WG16 [3 μg; (D)] or MARA3 [1:10 dilution; (E)] were analysed by immunoblotting (IB) with an anti-RecQ5β antibody or anti-RNAP II antibody [N-20 (C), 8WG16 (D) and MARA3 (E)]. As a control, immunoprecpitation was also performed using mouse or rabbit IgG antibody (IgG) and was analysed by immunoblotting. Cell lysates (50 μg) were also immunoblotted as a control (Input). (F and G) HEK-293 EBNA cells were transfected (TF) with the mock pcDNA 3.1 plasmid (−) or FLAG-tagged expression plasmids expressing RPB3 (F) or Pin1 (G) (+). The immunopreciptates isolated from transfected HEK-293 EBNA cell lysates (2000 μg) by FLAG immunoprecipitation (IP) were analysed by immunoblotting (IB) with an anti-RNAP II antibody (N-20), anti-RecQ5β antibody or anti-FLAG antibody. Cell lysates (50 μg) were also immunoblotted as a control (Input). (H) HEK-293 EBNA cells (2×106 cells) were fractionated into three fractions (Triton soluble fraction, Chromatin fraction and Nuclear matrix fraction) and 3% of each fraction was subjected to Western blot analysis with anti-RNAP II (8WG16), anti-RecQ5β, anti-PABP1, anti-hnRNP A1 and anti-lamin B antibodies. The Triton soluble fraction, chromatin fraction and nuclear matrix fraction were indicated by the detection of PABP1, hnRNP A1 and lamin B respectively. IB, immunoblotting.

To confirm the association of RecQ5β with the RNAP II large subunit further, we carried out immunoprecipitation of RNAP II using three different antibodies against RNAP II (N-20, 8WG16 and MARA3), and RecQ5β was detected in all of the immunoprecipitates by immunoblot analysis with an anti-RecQ5β antibody (Figures 2C–2E); thus the in vivo association between RecQ5β and the RNAP II large subunit was confirmed reciprocally. In addition, we isolated the complex associated with FLAG–RPB3; one of the proteins was identified by PMF/MALDI-TOF–MS, and confirmed that FLAG–RPB3 was associated not only with the RNAP II large subunit, but also with RecQ5β (Figure 2F). Since RNAP IIO is known to interact with Pin1, a peptidyl prolyl cistrans isomerase [34], we also isolated a protein complex associated with FLAG–Pin1 and showed that the FLAG–Pin1-associated complexes contained not only RNAP IIO, but also RecQ5β (Figure 2G), indicating that RecQ5β is associated with RNAP IIO as well as Pin1. Furthermore, RecQ5β was also detected in the chromatin fraction (Figure 2H), suggesting that RecQ5β is a constituent of the RNAP II transcriptional complex.

Sucrose-gradient fractionation of the RecQ5β-associated complex

We fractionated HEK-293 EBNA crude cell lysates into five fractions by sucrose-density-gradient ultracentrifugation and immunoprecipitated each fraction with the anti-RecQ5β antibody (Figure 3A). Although the anti-RecQ5β antibody immuno-precipitated only RecQ5β but not RNAP IIA in fraction one, it did precipitate both RecQ5β and RNAP IIA in fraction two, and, to a lesser extent, in fractions three and four. This result indicates that a subpopulation of RecQ5β is associated with RNAP IIA in the cell. The location of the RecQ5β–RNAP IIA complex in fraction two of the sucrose gradient also indicates that these proteins are present in a relatively large complex. It seems that the size of the major RecQ5β-associated complexes containing RNAP IIA is larger than that of the general splicing factor U2AF65-containing spliceosome, but smaller than that of the H-complex including hnRNP C1/C2 (Figure 3A) [35,36]. To determine the proportion of RNAP IIA associated with RecQ5β compared with the total 8WG16-detected RNAP IIA present in the cell extract, we performed immunodepletion of RecQ5β-associated complexes from cell extracts and found that more than 90% of RNAP IIA was not immunodepleted from cell extracts with RecQ5β (Figure 3B), suggesting that no more than 10% of the RNAP IIA population interacted with a subpopulation of RecQ5β.

Relative size of the RecQ5β-associated complex containing RNAP II

Figure 3
Relative size of the RecQ5β-associated complex containing RNAP II

(A) The RNPs-containing fraction obtained from HEK-293 EBNA cell lysates (2000 μg) was separated into ten fractions by ultracentrifugation on a 10–40% (v/v) sucrose gradient. UV absorption and the location of the 40 S, 60 S and 80 S ribosomal subunits indicated above their corresponding peak are shown (top panel). Two consecutive fractions were combined as indicated in the Figure and each of the combined fractions was subjected to immunoblot (IB) analysis using the anti-RNAP II antibody (8WG16), anti-hnRNP C1/C2 and anti-U2AF65 antibody. Cell lysates (50 μg) were also immunblotted as a control (Input) (middle panel). In addition, each of the combined fractions was subjected to immunoprecipitation (IP) with an anti-RecQ5β antibody and then subjected to immunoblot analysis using anti-RNAP II (8WG16) and anti-RecQ5β antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input) (bottom panel). (B) HEK-293 EBNA whole-cell lysates immunodepleted (ID) by unspecified IgG1 antibody (WCE) or by anti-RecQ5β antibody (ID-WCE) were analysed by Western blot analysis with anti-RNAP II (8WG16), anti-RecQ5β and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies. The histogram shows the relative amount of 8WG16-detected RNAP IIA present in the RecQ5β-depleted cell extract compared with that present in the cell lysates immunodepleted with the unspecified IgG1 antibody. The values were obtained by densitometry and were adjusted to those of GAPDH. Results are means±S.D. (n=4). *P<0.05 as determined by the Student's t test.

Figure 3
Relative size of the RecQ5β-associated complex containing RNAP II

(A) The RNPs-containing fraction obtained from HEK-293 EBNA cell lysates (2000 μg) was separated into ten fractions by ultracentrifugation on a 10–40% (v/v) sucrose gradient. UV absorption and the location of the 40 S, 60 S and 80 S ribosomal subunits indicated above their corresponding peak are shown (top panel). Two consecutive fractions were combined as indicated in the Figure and each of the combined fractions was subjected to immunoblot (IB) analysis using the anti-RNAP II antibody (8WG16), anti-hnRNP C1/C2 and anti-U2AF65 antibody. Cell lysates (50 μg) were also immunblotted as a control (Input) (middle panel). In addition, each of the combined fractions was subjected to immunoprecipitation (IP) with an anti-RecQ5β antibody and then subjected to immunoblot analysis using anti-RNAP II (8WG16) and anti-RecQ5β antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input) (bottom panel). (B) HEK-293 EBNA whole-cell lysates immunodepleted (ID) by unspecified IgG1 antibody (WCE) or by anti-RecQ5β antibody (ID-WCE) were analysed by Western blot analysis with anti-RNAP II (8WG16), anti-RecQ5β and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies. The histogram shows the relative amount of 8WG16-detected RNAP IIA present in the RecQ5β-depleted cell extract compared with that present in the cell lysates immunodepleted with the unspecified IgG1 antibody. The values were obtained by densitometry and were adjusted to those of GAPDH. Results are means±S.D. (n=4). *P<0.05 as determined by the Student's t test.

RNAP IIA and RNAP IIO associate with different domains of RecQ5β

To ascertain which fragment of RecQ5β interacts with the RNAP II large subunit, we constructed expression plasmids for full-length FLAG–RecQ5β and five initial truncation mutants containing either the natural RecQ5β localization signal or the SV40 large T-antigen NLS (β917, β744, β617, β533 and β395; see Figure 5A for details). The cellular localization (Figure 4A), association with RNAP II (Figures 4B and 4C) and the associated complex size (Figure 4D) of FLAG–RecQ5β and endogenous RecQ5β were indistinguishable. The five truncation mutants of FLAG–RecQ5β were also expressed (Figure 5A), and their predominantly nucleoplasmic localizations (Figure 5B) and molecular masses (Figure 5C) were confirmed. Protein complexes associated with each of the truncated mutants were immunoprecipitated using an anti-FLAG antibody and the samples were then subjected to immunoblot analysis using the N-20 and MARA3 antibodies. This analysis confirmed the association of both RNAP IIA and RNAP IIO with three of the truncation mutants (β917, β744 and β617), but not with the other two mutants (β533 and β395) (Figure 5C), suggesting that amino acid residues 534–617 of RecQ5β can interact with RNAP IIA and RNAP IIO. Four additional FLAG–RecQ5β truncation mutant expression plasmids containing the SV40 large T-antigen NLS were constructed (βc1, βc2, βc3 and βc4; see Figure 5A for details). Although all of these mutants located to the nucleoplasm (Figure 5B), only βc1 immunoprecipitated RNAP IIO (Figure 5D). The result seemed to be contradictory to the results described above. Therefore we constructed two additional truncation mutants (βc34 and βdHE; see Figure 5A) and examined the possibility that the full interaction with the RNAP II subunit requires amino acid residues located in a region other than amino acid residues 534–617 in the RecQ5β molecule (Figure 5E). We found that both RNAP IIA and RNAP IIO were associated with βc34, and showed that RNAP IIO was associated more strongly with βdHE than with βc34, as revealed by immunoblotting with both N-20 and MARA3 antibodies (Figure 5E). This suggests that the region containing amino acid residues 396–617 of the RecQ5β molecule is responsible for the association with both RNAP IIA and RNAP IIO; however, the association with RNAP IIO can occur within the region of amino acid residues 745–991 of RecQ5β (corresponding to the βc1 truncation mutant construct) independently of the interaction with the region containing amino acid residues 396–617. Thus RecQ5β associates with RNAP IIA and RNAP IIO through the distinctly different regions in the non-helicase domain of the RecQ5β molecule, suggesting that RecQ5β participates in processes associated with transcription, including those from pre-initiation to elongation [34].

Characterization of FLAG-RecQ5β via immunoprecipitation, cellular localization and sucrose-gradient analyses

Figure 4
Characterization of FLAG-RecQ5β via immunoprecipitation, cellular localization and sucrose-gradient analyses

(A) Immunofluorescence analysis of HEK-293 EBNA cells transfected with or without the FLAG-RecQ5β expression plasmid. Cells were treated with mouse anti-FLAG or anti-RecQ5β antibodies as the primary antibody respectively, followed by an Alexa Fluor® 488-conjugated anti-mouse IgG antibody as the secondary antibody. The cells were also stained with DAPI (4′,6-diamidino-2-phenylindole) as a nuclear marker. (B) Cell lysates (2000 μg) obtained from HEK-293 EBNA cells transfected (TF) with mock pcDNA3.1 plasmid (−) or the FLAG–RecQ5β expression plasmid (+) were subjected to immunoprecipitation (IP) using the anti-FLAG antibody. The FLAG–RecQ5β-associated complexes were then analysed by immunoblotting (IB) with the anti-RNAP II antibody (N20) and the anti-FLAG antibody. Cell lysates (50 μg) were also immunoblotted as a control (Input). (C) HEK-293 EBNA cells transfected (TF) with FLAG–RecQ5β and were subjected to immunoprecipitation (IP) using the anti-FLAG antibody. The immunoprecipitates were treated with (+) or without (−) DNase I or RNase A as indicated, followed by immunoblot (IB) analysis using anti-RNAP II (N20) and anti-FLAG antibodies. Cell lysates (50 μg) were also immunoblotted as a control (Input). (D) The RNP-containing fraction derived from FLAG–RecQ5β-transfected HEK-293 EBNA cells (2000 μg) was separated into ten fractions by ultracentrifugation on a 10–40% (v/v) sucrose gradient. UV absorption and the absorption peaks derived from the 40 S, 60 S and 80 S ribosomal subunits are indicated above the corresponding peaks (top panel). The fractions were subjected to immunoblot (IB) analysis using the anti-hnRNP C1/C2 and anti-U2AF65 antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input) (middle panel). In addition, each of the fractions was subjected to immunoprecipitation (IP) with an anti-FLAG antibody and the immunoprecipitates were subjected to immunoblot analysis using the anti-RNAP II (8WG16) and anti-FLAG antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input).

Figure 4
Characterization of FLAG-RecQ5β via immunoprecipitation, cellular localization and sucrose-gradient analyses

(A) Immunofluorescence analysis of HEK-293 EBNA cells transfected with or without the FLAG-RecQ5β expression plasmid. Cells were treated with mouse anti-FLAG or anti-RecQ5β antibodies as the primary antibody respectively, followed by an Alexa Fluor® 488-conjugated anti-mouse IgG antibody as the secondary antibody. The cells were also stained with DAPI (4′,6-diamidino-2-phenylindole) as a nuclear marker. (B) Cell lysates (2000 μg) obtained from HEK-293 EBNA cells transfected (TF) with mock pcDNA3.1 plasmid (−) or the FLAG–RecQ5β expression plasmid (+) were subjected to immunoprecipitation (IP) using the anti-FLAG antibody. The FLAG–RecQ5β-associated complexes were then analysed by immunoblotting (IB) with the anti-RNAP II antibody (N20) and the anti-FLAG antibody. Cell lysates (50 μg) were also immunoblotted as a control (Input). (C) HEK-293 EBNA cells transfected (TF) with FLAG–RecQ5β and were subjected to immunoprecipitation (IP) using the anti-FLAG antibody. The immunoprecipitates were treated with (+) or without (−) DNase I or RNase A as indicated, followed by immunoblot (IB) analysis using anti-RNAP II (N20) and anti-FLAG antibodies. Cell lysates (50 μg) were also immunoblotted as a control (Input). (D) The RNP-containing fraction derived from FLAG–RecQ5β-transfected HEK-293 EBNA cells (2000 μg) was separated into ten fractions by ultracentrifugation on a 10–40% (v/v) sucrose gradient. UV absorption and the absorption peaks derived from the 40 S, 60 S and 80 S ribosomal subunits are indicated above the corresponding peaks (top panel). The fractions were subjected to immunoblot (IB) analysis using the anti-hnRNP C1/C2 and anti-U2AF65 antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input) (middle panel). In addition, each of the fractions was subjected to immunoprecipitation (IP) with an anti-FLAG antibody and the immunoprecipitates were subjected to immunoblot analysis using the anti-RNAP II (8WG16) and anti-FLAG antibodies. Cell lysates (50 μg) were also immunblotted as a control (Input).

Determination of the RNAP II-associated domain of RecQ5β by the use of truncation mutants

Figure 5
Determination of the RNAP II-associated domain of RecQ5β by the use of truncation mutants

(A) Schematic diagram of the full-length FLAG–RecQ5β and the truncation mutants. The N-terminal helicase domain (grey box); HCRs (highly conserved regions) 1 (left-hand side hatched box), 2 (right-hand side hatched box) and 3 (central hatched box) and the internal NLS (black box) are indicated. The C-terminal residue numbers for RecQ5β and the truncation mutants (β917, β744, β617, β533, β395, βc1, βc2, βc3, βc4, βc34 and βdHE) are indicated. A FLAG tag (black ovals) was added to the N-terminus of each peptide. For truncation mutants lacking the internal NLS of RecQ5β, the SV40 large T-antigen NLS (white ovals) was added to the C-terminus. (B) Each of the FLAG–tagged truncation mutants (β917, β744, β617, β533, β395, βc1, βc2, βc3, βc4, βc34 and βdHE) were transiently expressed in HEK-293 EBNA cells. The truncation mutants were detected using mouse anti-FLAG antibody as the primary antibody and stained with Alexa Fluor® 488-conjugated anti-mouse IgG antibody as the secondary antibody. The HEK-293 EBNA cells were also stained with DAPI (4′,6-diamidino-2-phenylindole) as a nuclear marker. (CE) HEK-293 EBNA cells were transfected (TF) with expression plasmids for full-length FLAG–RecQ5β or the truncated FLAG–RecQ5β mutants [β917, β744, β617, β533 and β395 (C); βc1, βc1, βc3 and βc4 (D); βc34 and βdHE (E)] and the FLAG-tagged protein-associated complexes were immunoprecipitated (IP) from cell lysates (2000 μg) using anti-FLAG M2 antibody–beads. The immunoprecipitates were then subjected to immunoblot (IB) analysis with anti-RNAP II N20 (CE), anti-RNAP II MARA3 (C and E) and anti-FLAG antibodies (CE). As a negative control, HEK-293 EBNA cells transfected with mock pcDNA3.1 plasmid were also used for immunoprecipitation (−). Cell lysates (50 μg) were also immunoblotted as a control (Input). Molecular-mass markers are indicated at the left-hand side (in kDa). Asterisks (*) indicate the position of each of the truncation mutants, which migrated according to their theoretical molecular mass calculated from the amino acid sequences.

Figure 5
Determination of the RNAP II-associated domain of RecQ5β by the use of truncation mutants

(A) Schematic diagram of the full-length FLAG–RecQ5β and the truncation mutants. The N-terminal helicase domain (grey box); HCRs (highly conserved regions) 1 (left-hand side hatched box), 2 (right-hand side hatched box) and 3 (central hatched box) and the internal NLS (black box) are indicated. The C-terminal residue numbers for RecQ5β and the truncation mutants (β917, β744, β617, β533, β395, βc1, βc2, βc3, βc4, βc34 and βdHE) are indicated. A FLAG tag (black ovals) was added to the N-terminus of each peptide. For truncation mutants lacking the internal NLS of RecQ5β, the SV40 large T-antigen NLS (white ovals) was added to the C-terminus. (B) Each of the FLAG–tagged truncation mutants (β917, β744, β617, β533, β395, βc1, βc2, βc3, βc4, βc34 and βdHE) were transiently expressed in HEK-293 EBNA cells. The truncation mutants were detected using mouse anti-FLAG antibody as the primary antibody and stained with Alexa Fluor® 488-conjugated anti-mouse IgG antibody as the secondary antibody. The HEK-293 EBNA cells were also stained with DAPI (4′,6-diamidino-2-phenylindole) as a nuclear marker. (CE) HEK-293 EBNA cells were transfected (TF) with expression plasmids for full-length FLAG–RecQ5β or the truncated FLAG–RecQ5β mutants [β917, β744, β617, β533 and β395 (C); βc1, βc1, βc3 and βc4 (D); βc34 and βdHE (E)] and the FLAG-tagged protein-associated complexes were immunoprecipitated (IP) from cell lysates (2000 μg) using anti-FLAG M2 antibody–beads. The immunoprecipitates were then subjected to immunoblot (IB) analysis with anti-RNAP II N20 (CE), anti-RNAP II MARA3 (C and E) and anti-FLAG antibodies (CE). As a negative control, HEK-293 EBNA cells transfected with mock pcDNA3.1 plasmid were also used for immunoprecipitation (−). Cell lysates (50 μg) were also immunoblotted as a control (Input). Molecular-mass markers are indicated at the left-hand side (in kDa). Asterisks (*) indicate the position of each of the truncation mutants, which migrated according to their theoretical molecular mass calculated from the amino acid sequences.

Because the region of amino acid residues 396–617 is responsible for binding to both forms of RNAP, and the region containing amino acid residues 745–991 is responsible for the interaction with RNAP IIO, the RNAP II large subunit may be transferred from one site to the other on the RecQ5β molecule, possibly induced by phosphorylation/dephosphorylation of the RNAP II large subunit during transcription and/or its related processes, such as those which occur as a result of sudden transcriptional inhibition etc. In fact, immunoprecipitation of the RecQ5β-associated complexes prepared after actinomycin D treatment of HEK-293 EBNA cells followed by immunoblot analysis indicated that actinomycin D increased the association of RecQ5β with RNAP IIO, whereas it decreased the association with RNAP IIA (Figure 6). This biochemical feature is very intriguing, because it may connect the known enzymatic activity of RecQ5β as an ssDNA-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities [21] to the proposed role of RecQ5β in transcription. Namely, the events caused by the helicase activity may be coupled closely to the transfer of RNAP IIA or RNAP IIO on to the RecQ5β molecule, which is possibly required for the regulation of transcription and/or related processes.

Characterization of the interaction between RecQ5β and RNAP II upon the treatment of transcription inhibitor

Figure 6
Characterization of the interaction between RecQ5β and RNAP II upon the treatment of transcription inhibitor

HEK-293 EBNA cells were incubated with (+) or without (−) 5 μg/ml actinomycin D (act D) for 3 h, and subjected to immunoprecipitation (IP) with an anti-RecQ5β antibody. The immunoprecipitates from 1 μg of cell lysates were analysed by immunoblotting (IB)with anti-RNAP II (N-20) and anti-RecQ5β antibodies. Cell lysates (50 μg) were also immunoblotted as a control (Input).

Figure 6
Characterization of the interaction between RecQ5β and RNAP II upon the treatment of transcription inhibitor

HEK-293 EBNA cells were incubated with (+) or without (−) 5 μg/ml actinomycin D (act D) for 3 h, and subjected to immunoprecipitation (IP) with an anti-RecQ5β antibody. The immunoprecipitates from 1 μg of cell lysates were analysed by immunoblotting (IB)with anti-RNAP II (N-20) and anti-RecQ5β antibodies. Cell lysates (50 μg) were also immunoblotted as a control (Input).

RecQ5β associates with coding regions of the genes examined

To examine whether RecQ5β associates with some active genes further, we randomly selected two genes (the LDL receptor and β-actin genes) and performed ChIP with FLAG–RecQ5β-expressing cells. We found that FLAG–RecQ5β associated mostly with the coding regions of the two genes examined, but not with the promoter regions (Figures 7A–7C). This result suggests strongly that RecQ5β associates with active genes, and is consistent with the observation that RecQ5β associates not only with RNAP IIA, but also with RNAP IIO, which is involved in active transcription. Finally, we examined the effect of knockdown of the RecQ5β transcript with siRNA on the transcription of the LDL receptor and β-actin genes and found that knockdown of RecQ5β increased the mRNA levels of LDL receptor and β-actin 2.2- and 1.3-fold respectively when 7SK RNA was used as control (Figures 7D and 7E). Although we cannot exclude at present the possibility that RecQ5β accelerates destabilization of those mRNAs, these results suggest that RecQ5β plays an inhibitory role in transcription of at least some specific genes. Because RecQ5β is associated with both RNAP IIA and RNAP IIO, RecQ5β may provide a temporal docking site for those proteins during ongoing transcription and/or related processes.

ChIP analysis of the RecQ5β-associated genes and RNAi analysis of the RecQ5β transcript

Figure 7
ChIP analysis of the RecQ5β-associated genes and RNAi analysis of the RecQ5β transcript

(A) Cell lysates from FLAG–RecQ5β-transfected HEK-293 EBNA cells (5×104 cells) were analysed by immunoblotting (IB) with an anti-FLAG antibody. Tubulin β was also immunoblotted as a loading control. Mock, cell lysates obtained from the mock vector-transfected HEK-293 EBNA cells. (B) The result of a typical ChIP assay is shown. Cell lysates from FLAG–RecQ5β-transfected (TF) HEK-293 EBNA cells was subjected to ChIP using an anti-FLAG antibody (α-FLAG), anti-RNAP II antibody (N-20, positive control) or unspecified rabbit polyclonal IgG (IgG, negative control). The target DNA fragments, LDL receptor (LDLR) promoter and coding region, β-actin (ActB) promoter and coding region and mitochondrial DNA (mtDNA) were amplified by 27–33 cycles of PCR and then separated by agarose gel electrophoresis and stained with ethidium bromide. The relative intensity of each amplified target DNA fragment associated with anti-FLAG or IgG antibodies is shown at the bottom of each panel. Input, DNA fragments extracted from 1% of the cell lysates. (C) ChIP experiments were performed for each target DNA fragment. The values obtained for unspecified IgG were used to normalize the intensities of the amplified target DNA fragments indicated. Results are means±S.D. (n=3) and P values are also indicated. TF, transfected. (D) HeLa cells were transfected with siRNA or scRNA oligonucleotides for 96 h. Immunoblot (IB) analysis was performed using anti-RecQ5β and anti-tubulin β antibodies. (E) The levels of LDL receptor (LDLR) or β-actin (ActB) mRNAs were assessed by quantitative real-time PCR. The level of 7SK RNA mRNA was used as an internal control. Results are means±S.D. (n=3) and P values are indicated.

Figure 7
ChIP analysis of the RecQ5β-associated genes and RNAi analysis of the RecQ5β transcript

(A) Cell lysates from FLAG–RecQ5β-transfected HEK-293 EBNA cells (5×104 cells) were analysed by immunoblotting (IB) with an anti-FLAG antibody. Tubulin β was also immunoblotted as a loading control. Mock, cell lysates obtained from the mock vector-transfected HEK-293 EBNA cells. (B) The result of a typical ChIP assay is shown. Cell lysates from FLAG–RecQ5β-transfected (TF) HEK-293 EBNA cells was subjected to ChIP using an anti-FLAG antibody (α-FLAG), anti-RNAP II antibody (N-20, positive control) or unspecified rabbit polyclonal IgG (IgG, negative control). The target DNA fragments, LDL receptor (LDLR) promoter and coding region, β-actin (ActB) promoter and coding region and mitochondrial DNA (mtDNA) were amplified by 27–33 cycles of PCR and then separated by agarose gel electrophoresis and stained with ethidium bromide. The relative intensity of each amplified target DNA fragment associated with anti-FLAG or IgG antibodies is shown at the bottom of each panel. Input, DNA fragments extracted from 1% of the cell lysates. (C) ChIP experiments were performed for each target DNA fragment. The values obtained for unspecified IgG were used to normalize the intensities of the amplified target DNA fragments indicated. Results are means±S.D. (n=3) and P values are also indicated. TF, transfected. (D) HeLa cells were transfected with siRNA or scRNA oligonucleotides for 96 h. Immunoblot (IB) analysis was performed using anti-RecQ5β and anti-tubulin β antibodies. (E) The levels of LDL receptor (LDLR) or β-actin (ActB) mRNAs were assessed by quantitative real-time PCR. The level of 7SK RNA mRNA was used as an internal control. Results are means±S.D. (n=3) and P values are indicated.

We thank Dr Bart Sefton (Molecular and Cell Biology Laboratory, The Salk Institute, La Jolla, CA, U.S.A) for providing the MARA3 antibody. This work was supported in part by a grant from Pioneer Research on the Genome the Ministry of Education, Culture, Sports, Science and Technology of Japan and also in part by a Grant-in-Aid for science research from the JSPS (Japan Society for the Promotion of Science).

Abbreviations

     
  • BLM

    Bloom syndrome helicase

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DTT

    dithiothreitol

  •  
  • EBNA

    Epstein–Barr virus nuclear antigen

  •  
  • HEK-293

    human embryonic kidney-293

  •  
  • LDL

    low-density lipoprotein

  •  
  • MALDI-TOF–MS

    matrix-assisted laser-desorption ionization–time-of-flight MS

  •  
  • NLS

    nuclear localization signal

  •  
  • PABP1

    poly-A binding protein 1

  •  
  • PBST

    PBS containing 0.05% Tween 20

  •  
  • PMF

    peptide mass fingerprinting

  •  
  • RNAi

    RNA interference

  •  
  • RNAP II

    RNA polymerase II

  •  
  • RPB3

    RNAP II 33 kDa subunit

  •  
  • RNAP IIA

    hypophosphorylated RNAP II large subunit

  •  
  • RNAP IIO

    hyperphosphorylated RNAP II large subunit

  •  
  • RNP

    ribonucleoprotein

  •  
  • hnRNP

    heterogeneous nuclear RNP

  •  
  • scRNA

    scrambled control RNA

  •  
  • siRNA

    small interfering RNA

  •  
  • ssDNA

    single-stranded DNA

  •  
  • SV40

    simian virus 40

  •  
  • TBS

    Tris-buffered saline

  •  
  • TBST

    TBS containing 0.1% Tween 20

  •  
  • WRN

    Werner syndrome helicase

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Author notes

1

Present address: Institute for Environmental and Gender-specific medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279–0021, Japan

2

Present address: Department of Bioscience and Bioinformatics, College of Information Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu 525–0058, Japan

3

Present address: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Hongo, Bunkyo-ku, Tokyo 113–8657, Japan

4

Present address: Department of Hygiene, Dokkyo University School of Medicine, 180 Kitakobayashi, Mibu, Tochigi 321–0293, Japan

5

Present address: Department of Cellular and Molecular Biology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734–8553, Japan

Supplementary data