Actin, the major component of the cytoplasmic skeleton, has been shown to exist in the nucleus. Nuclear actin functions in several steps of the transcription process, including chromatin remodelling and transcription initiation and elongation. However, as a part of PICs (pre-initiation complexes), the role of actin remains to be elucidated. In the present study, we identified RHA (RNA helicase A) as an actin-interacting protein in PICs. Using immunoprecipitation and immunofluorescence techniques, we have shown that RHA associates with β-actin in the nucleus. A GST (glutathione transferase) pulldown assay using different deletion mutants revealed that the RGG (Arg-Gly-Gly) region of RHA was responsible for the interaction with β-actin, and this dominant-negative mutant reduced the recruitment of Pol II (RNA polymerase II) into PICs. Moreover, overexpression or depletion of RHA could influence the interaction of Pol II with β-actin and β-actin-involved gene transcription regulation. These results suggest that RHA acts as a bridging factor linking nuclear β-actin with Pol II.
- nuclear β-actin
- pre-initiation complex
- RGG (Arg-Gly-Gly) region
- RNA helicase A
- RNA polymerase II
In eukaryotes, transcription is a strictly regulated process . First, the condensed chromatin is activated by the chromatin remodelling complex. Then Pol II (RNA polymerase II) and GTFs (general transcription factors) are recruited to promoters by gene-specific transcription factors to form the PIC (pre-initiation complex). Ultimately, these large transcriptional complexes proceed through initiation, promoter clearance, elongation and termination. Recently, nuclear actin has been shown to be connected with many of these transcriptional events. First, actin is a component of the chromatin remodelling complex and is required for its function in transcription activity . Secondly, actin interacts with Pol I, II and III, and plays a key role in basal transcription [3–7]. Thirdly, actin associates with hnRNP (heterogeneous nuclear ribonucleoprotein) and functions as a platform for the recruitment of HAT (histone acetyltransferase) or HDAC (histone deacetylase) complexes along the active transcript unit [8,9].
Transcription begins with the assembly of PICs, and formation of this complex is assisted by various transcriptional activators, cofactors and mediators . Acting as a cofactor, β-actin was identified as a component of Pol II PICs, and antibodies against β-actin inhibited Pol II transcription and the recruitment of Pol II into PICs . However, the role of actin in this complex remains unclear. It has been reported that RHA (RNA helicase A) binds with filamentous actin  and also purified Pol II , raising the possibility that RHA acts as a bridging factor linking actin with Pol II.
RHA is a member of the DExH family of ATPase/helicase, which contains two dsRBDs (double-stranded RNA-binding domains), a catalytic core domain and an RGG (Arg-Gly-Gly) box . RHA catalyses both duplex RNA and DNA in an ATP-dependent manner. RHA was originally isolated as a human homologue of the Drosophila MLE (maleless) protein, which is involved in dosage compensation by increasing the transcription activity of X-linked genes in males . Similar to MLE, RHA plays many roles in transcriptional regulation. RHA acted as a bridging factor, coupling Pol II to the transcriptional coactivator CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300  and BRCA1 (breast-cancer susceptibility gene 1) . RHA was also reported to be involved in HIV gene expression  and transcription regulation of p16INK4a . In addition, RHA interacted with NF-κB (nuclear factor κB) p65 and enhanced its transcriptional activity . These reports suggest that RHA is an essential factor for a wide variety of transcription pathways.
In the present study, we identified RHA as an actin-interacting protein in PICs. RHA interacted with β-actin via the RGG region, and disruption of the interaction by using an RGG fragment reduced the recruitment of Pol II to the PIC. Overexpression or depletion of RHA influenced the interaction of Pol II with β-actin and β-actin-involved gene transcription regulation. These results suggest that RHA plays an important role in linking β-actin with Pol II and functions in gene transcription regulation.
Cell culture and transfection
HeLa and HEK (human embryonic kidney)-293T cells were maintained in IMDM (Iscove's modified Dulbecco's medium) supplemented with 10% FCS (fetal calf serum), 100 units/ml penicillin and 100 μg/ml streptomycin. The cells were grown at 37 °C in a humidified 95% air, 5% CO2 atmosphere. HeLa cells were transfected with Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol, and HEK-293T cells were transfected using a standard calcium phosphate method.
HA (haemagglutinin)-tagged RHA and K417E mutant were generously provided by Dr Toshihiro Nakajima (Department of Genome Science, Institute of Medical Science, St. Marianna University School of Medicine, Japan). Fragments of RHA in pGEX were gifts from Dr Jeffrey Parvin (Harvard Medical School, Boston, MA, U.S.A.). pREP7-Rluc vector was kindly provided by Dr Keji Zhao (National Institutes of Health, Bethesda, MD, U.S.A.). IL-8-luc (where IL is interleukin and luc is luciferase) reporter plasmid was kindly supplied by Dr Charalabos Pothoulakis (Harvard Medical School, Boston, MA, U.S.A.). pBluescript II-KS(+)-AdMLP (where AdMLP is adenovirus major late promoter) was a gift from Dr Wilma Hofmann (Department of Physiology and Biophysics, University of Illinois at Chicago, IL, Chicago, U.S.A.). To create HA-tagged, FLAG-tagged and His-tagged β-actin expression plasmid, the β-actin fragment was amplified by PCR and subcloned in-frame into pcDNA3-HA, pcDNA3-FLAG and pET-28a vector respectively. The β-actin deletion mutants fused to GST (glutathione transferase) were generated by PCR and introduced into the pGEX-6p-1 vector (Amersham Pharmacia Biotech). The CSF-1-luc (where CSF-1 is colony-stimulating factor-1) reporter plasmid was constructed by inserting the CSF-1 promoter (containing −567 to +80) into the KpnI/HindIII sites of pGL3 vector. All of the PCR-generated plasmids were confirmed by sequence analysis.
Antibodies and reagents
Rabbit anti-HA antibody (Y-11) and anti-Pol II antibody (N-20) were from Santa Cruz Biotechnology. Rabbit anti-RHA antibody (ab26271) was from Abcam. Mouse anti-β-actin antibody (A5441), anti-FLAG M2 antibody (F1804) and anti-β-tubulin antibody (T4026) were from Sigma–Aldrich. HRP (horseradish peroxidase)-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, TRITC (tetramethylrhodamine β-isothiocyanate)-conjugated anti-mouse IgG, and FITC-conjugated anti-rabbit IgG were from The Jackson Laboratory. ECL (enhanced chemiluminescence) Plus Western blotting detection reagents (RPN2132) and glutathione–Sepharose 4B (17-0756-01) were purchased from Amersham Biosciences. Ni-NTA (Ni2+-nitrilotriacetate) agarose (1018244) was bought from Qiagen.
Nuclear extraction was performed as described previously [19,20]. Briefly, cells were washed with PBS, then lysed in a lysis buffer [20 mM Hepes (pH 7.2), 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P40, 1 mM Na3VO4, 1 mM PMSF and 10 mg/ml aprotinin] and homogenized by a tightly fitting Dounce homogenizer. The homogenate was centrifuged at 1500 g for 5 min to pellet the nuclei. The nuclear pellet was washed three times with lysis buffer and resuspended in the same lysis buffer supplemented with 0.5 M NaCl to extract nuclear proteins. The extracted material was sedimented at 15000 g for 10 min, and the resulting supernatant was harvested as the nuclear extracts. The protein concentration was determined using the Bradford assay.
Immunoprecipitation and immunoblotting
Immunoprecipitation was carried out using nuclear extracts and the appropriate antibodies as indicated, at 4 °C for 3 h, followed by incubation for another 3 h with Protein A/G–Sepharose. After washing with Nonidet P40 lysis buffer [20 mM Tris/HCl (pH 8.0), 137 mM NaCl, 1% Nonidet P40, 10% glycerol, 1 mM Na3VO4, 1 mM PMSF, 10 mg/ml aprotinin and 20 mg/ml leupeptin], immunoprecipitates were resolved on SDS/PAGE, electroblotted on to nitrocellulose membranes (Millipore) and probed with antibodies as indicated. Chemiluminescent detection was performed by using ECL plus Western blotting reagents.
HeLa cells on coverslips were washed once with PBS, fixed in 4% (w/v) paraformaldehyde in PBS for 20 min and then permeabilized by 0.5% Triton X-100 in PBS for 30 min. Non-specific binding was prevented by the addition of 10% (v/v) bovine serum in PBS for 30 min. Then the cells were incubated for 1 h with anti-β-actin and anti-RHA antibodies. After washing with PBS, the cells were incubated for 1 h with TRITC-conjugated secondary antibody against mouse IgG and FITC-conjugated secondary antibody against rabbit IgG. After two washes with PBS, the nuclei were stained with Hoechst (DNA staining). After a final washing step with PBS, the coverslips were placed on to glass slides with mounting medium. The cells were observed using a laser-scanning confocal microscope (FluoView FV1000; Olympus) equipped with a 60×oil-immersion objective lens (UPLSAPO, Olympus; NA 1.35). Fluorescent images were collected using Olympus FV10-ASW 1.7 software and processed using Adobe Photoshop.
Protein expression and purification
GST-fused proteins were expressed in Escherichia coli BL21 (DE3) by induction with 1 mM IPTG (isopropyl β-D-thiogalactoside) at 37 °C for 3 h. The cells were harvested and sonicated in lysis buffer [20 mM Hepes (pH 7.5), 120 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM PMSF and 10 μg/ml each of aprotinin and leupeptin]. After centrifugation (15000 g for 30 min at 4 °C), the supernatants were incubated with glutathione–Sepharose 4B at 4 °C for 2 h. The beads were precipitated, washed five times with lysis buffer and then eluted with elution buffer [20 mM reduced glutathione, 100 mM Tris/HCl (pH 8.0) and 120 mM NaCl] .
E. coli BL21 (DE3) transformed with pET-28a-β-actin were grown at 37 °C to a D600 ≈ 0.5 and were induced by the addition of 1 mM IPTG. Cells were further cultured for 2 h at room temperature (25 °C) in the presence of 20 μM ZnSO4. Lysates were prepared by sonication in lysis buffer [10 mM Tris/HCl (pH 8.0), 500 mM NaCl, 0.1% Tween 20, 10% glycerol, 10 mM 2-mercaptoethanol, 10 μM ZnSO4, 10 mM imidazole and 1 mM PMSF]. Triton X-100 was added to a concentration of 1% and extracts were clarified by centrifugation (15000 g for 15 min at 4 °C). The soluble extract was incubated at 4 °C with gentle rolling overnight with Ni-NTA resin. The resin was washed with buffer E [20 mM Hepes (pH 7.5), 100 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM DTT (dithiothreitol), 10 μM ZnSO4, 20 mM imidazole and 1 mM PMSF] and eluted with buffer E/200 mM imidazole.
GST pulldown assays
GST and GST-fused protein immobilized on 40 μl of glutathione–Sepharose 4B were incubated with nuclear extracts or purified protein on a rotator at 4 °C for 3 h. After being washed four times with Nonidet P40 lysis buffer for nuclear extracts, or modified GBT buffer [50 mM Hepes (pH 7.5), 175 mM KCl, 10% glycerol, 7.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT and 1% Triton X-100] for purified protein, the bound proteins were analysed by immunoblotting.
The PIC formation assay was performed as described previously . Briefly, biotinylated templates were prepared by PCR using a plasmid containing AdMLP fused to a 390-bp G-less cassette. The DNA template was bound to streptavidin–Sepharose beads (Dynal). PICs were assembled on the immobilized DNA template by incubating with HeLa nuclear extracts (Promega) in transcription buffer [10 mM Hepes, 100 mM K-glutamate, 2.5 mM MgCl2 and 3.5% glycerol (to pH 7.6 with KOH)] at 28 °C for 30 min. The beads were washed and eluted, then analysed by immunoblotting. As indicated, HeLa nuclear extracts were incubated with either buffer, antibody (4 μg) or purified protein (2 μg) on ice for 30 min before incubation with the immobilized DNA template.
RNAi (RNA interference)
The double-stranded RNA specific for RHA was synthesized by Shanghai GeneChem. This siRNA (small interference RNA), 5′-AAGAAGUGCAAGCGACUCUAGTT-3′, was targeted to RHA from 544 to 564 bp [22,23]. Control siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′ was purchased from GenePharma. siRNA transfections were performed using Lipofectamine™ 2000 according to the manufacturer's instructions. Briefly, HeLa cells were grown to 50–70% confluence in 24-well plates. Transfection was carried out with 20 pmol of siRNA duplex in the culture medium. At 24 h later, cells were either harvested for immunoprecipitation, or transfected again for dual-luciferase assays.
HeLa cells were transiently transfected with 500 ng of CSF-1 or IL-8 reporter plasmid, 20 ng of pREP7-Rluc control plasmid or 500 ng of β-actin and/or RHA expression plasmid. The total amount of expression vector was kept constant by adding appropriate amounts of the empty vector pcDNA3-HA. The relative luciferase activity was analysed after 24 h using a Turner Designs TD 20/20 luminometer with the dual-luciferase assay system (Promega). All experiments were performed in triplicate, and all results were obtained from at least three separate experiments.
RHA and β-actin are present in PICs and associate with each other
Although β-actin is a component of PICs, the molecular mechanism by which actin affects transcription regulation remains unclear. RHA binds actin and Pol II, therefore we hypothesize that RHA plays a role in the function of β-actin in PICs. To explore this possibility, we first examined whether RHA existed in PICs by performing a PIC formation assay as described in the Experimental section. As shown in Figure 1(A), RHA and β-actin, as well as Pol II, were found in PICs, and β-tubulin could not be detected (used as a negative control).
Having demonstrated that RHA is a component of PICs, the interaction between RHA and β-actin was next analysed by immunoprecipitation. RHA and β-actin were immunoprecipitated from HeLa nuclear extracts, and the immunoprecipitates were analysed by Western blotting using antibodies against Pol II, RHA and β-actin. As shown in Figures 1(B) and 1(C), β-actin and Pol II co-immunoprecipitated with RHA, and RHA and Pol II co-immunoprecipitated with β-actin, indicating that Pol II, RHA and β-actin are in the same complex in the nucleus.
To validate the association of RHA with β-actin, HEK-293T cells were co-transfected with HA-tagged RHA (HA–RHA) and FLAG-tagged β-actin (FLAG–β-actin), and immunoprecipitation from nuclear extracts was performed using either IgG, an anti-HA antibody or an anti-FLAG antibody. Western blot analysis showed that RHA and β-actin were present in the samples immunoprecipitated with anti-HA and anti-FLAG antibodies, but not in the control sample (Figure 1D).
To directly test whether RHA and β-actin are co-localized in the nucleus, we performed an immunofluorescence double-labelling experiment. As shown in Figure 1(E), the labelling for RHA was restricted to the nucleus, whereas the labelling for β-actin was more extensive and distributed throughout the cell. Yellow dots in the merged image show the co-localization of RHA and β-actin. These results demonstrate that RHA associates with β-actin in the nucleus.
RHA interacts with the N-terminal of β-actin via the RGG region
To identify the domains involved in the RHA–β-actin interaction, a GST pulldown assay was performed. We constructed β-actin deletion mutants (A1–A3) as shown in Figure 2(A), and these GST-fused β-actin deletion mutants and GST alone were incubated with HeLa nuclear extracts. After washing, the bound proteins were analysed by immunoblotting with an antibody against RHA. Figure 2(B) shows that RHA bound strongly to the A1 deletion mutant, poorly to the A2 deletion mutant, and failed to bind to the A3 deletion mutant. We also performed a GST pulldown assay using GST-fused RHA deletion mutants (R1–R5, Figure 2C). As shown in Figure 2(D), β-actin could only bind to R5, but not the other deletion mutants.
While comparing with the actin-binding motif, we identified two potential actin-binding motifs in the RGG region of the R5 mutant (Figure 2E), which were similar to the myosin II head for binding to actin . We then used the RGG region (amino acid residues 1150–1279) for detecting the direct interaction with β-actin by using an in vitro protein-binding assay. Figure 2(F) showed that the RGG region, but not GST alone, could bind the purified β-actin. Taken together, these results indicate that the RGG region is responsible for the interaction of RHA with β-actin.
Disruption of the interaction between RHA and β-actin reduces the recruitment of Pol II into PICs
We have shown that RHA could interact with β-actin In vivo and in vitro. Next we examined the functions of these two proteins in PICs. PICs were assembled on the immobilized DNA templates by incubating with the HeLa nuclear extracts, which were pre-incubated with buffer, or with the anti-β-actin antibody or anti-RHA antibody. The assembled PICs were washed, eluted from the beads and analysed by immunoblotting with antibodies against Pol II, RHA and β-actin. As shown in Figure 3(A), the anti-β-actin antibody and anti-RHA antibody could both decrease the amounts of Pol II bound to the promoter, indicating that β-actin and RHA are required for the recruitment of Pol II into PICs.
To investigate the functional importance of the RHA–β-actin interaction in PICs, we examined the effect of the potential dominant-negative mutant RGG on the formation of PICs. PICs were assembled on the templates by incubating with the HeLa nuclear extracts, which were pre-incubated with GST or GST–RGG. As shown in Figure 3(B), GST–RGG significantly reduced the binding of β-actin, RHA and Pol II to the promoter, compared with the GST alone. Because RGG is a specific β-actin-binding domain in RHA, our results suggest that the RHA–β-actin interaction is important for the recruitment of Pol II into PICs.
RHA acts as a bridging factor between Pol II and β-actin
RHA could act as a bridging factor, coupling Pol II with CBP/p300 and BRCA1. Therefore we were interested in determining whether RHA may function as an adaptor to link β-actin with Pol II. Nuclear extracts were prepared from HEK-293T cells transfected with a plasmid encoding HA–RHA or HA vector. The samples were immunoprecipitated with anti-β-actin antibody, and the immunoprecipitates were analysed by Western blotting. As expected, Pol II could interact with β-actin (Figure 4A, lane 3), and this interaction was enhanced due to the overexpression of RHA (lane 2), whereas almost no interaction occurred in the control sample (lane 1). To verify this result, GST and GST–β-actin were incubated with the nuclear extracts as described above, and the bound proteins were analysed by immunoblotting. As shown in Figure 4(B), GST–β-actin could pulldown a higher amount of Pol II, especially the initiation form of Pol II (Pol IIA) in the samples expressing HA–RHA, compared with the samples expressing HA-vector, whereas no signal was observed in the GST control. These results suggest that RHA overexpression enhances the interaction between β-actin and Pol II.
Next, we wanted to explore the effect of RHA depletion on the interaction between β-actin and Pol II. We prepared an siRNA that could specifically reduce the endogenous RHA protein level (Figure 4C). Immunoprecipitation was performed using the nuclear extracts of HeLa cells transfected with control siRNA or RHA siRNA. As shown in Figure 4(D), knockdown of endogenous RHA significantly reduced the interaction of Pol II with β-actin. Similar results were obtained from the GST-pulldown assay (Figure 4E). Taken together, these results have presented a role for RHA in linking Pol II with β-actin.
RHA functions in β-actin-involved gene transcription regulation
The above results have shown that RHA is associated with β-actin and might affect gene transcription regulation. To gain further evidence to verify this possibility, CSF-1, which can be regulated by nuclear actin [25,26], was chosen as a target gene. HeLa cells were co-transfected with the CSF-1 reporter plasmid, pREP7-Rluc control plasmid, β-actin and RHA expression plasmids. As shown in Figure 5(A), β-actin alone promoted the luciferase activity approx. 2-fold (lane 2), and RHA alone did not significantly enhance the luciferase activity (lane 3). The co-expression of β-actin and RHA resulted in a 3-fold stimulation of the luciferase activity (lane 4), indicating that RHA can co-operate with β-actin to activate the CSF-1 promoter. Next, to investigate whether catalytic activity of RHA is required for the activation of β-actin-involved CSF-1 transcription regulation, cells were transfected with the CSF-1 reporter plasmid, pREP7-Rluc control plasmid, β-actin expression plasmid and RHA mutant K417E that is defective in nucleotide binding. As shown in Figure 5(A) (lane 5), the mutant was as effective as wild-type RHA, indicating that the catalytic activity of RHA is dispensable for the activation of β-actin-involved CSF-1 transcription regulation.
We then examined the effect of RHA depletion on β-actin-involved gene transcription regulation. As shown in Figure 5(B), RHA depletion reduced the β-actin-activated CSF-1 transcription regulation by 45% compared with the control. Similarly, RHA depletion also reduced IL-8 (another β-actin-regulated gene; results not shown) transcription regulation by 58% (Figure 5C). Taken together, these results suggest that RHA plays an important role in β-actin-involved gene transcription regulation.
Actin is an abundant protein in the cytoplasm, and plays a crucial role in cell morphology, motility and cytokinesis. Accumulating results have shown that actin also exists in the nucleus [27,28]. Although the cytoplasmic functions of actin are well understood, the roles of actin in the nucleus are controversial, especially the molecular mechanisms of actin in transcription regulation [29,30]. A previous study has demonstrated that β-actin is a component of PICs; however, the role of actin in this complex remains unclear . PIC formation occurs by stepwise recruitment of TBP (TATA-box-binding protein), TFIIB (transcription factor IIB), Pol II–TFIIF, TFIIE and TFIIH . In vivo, this process is more complicated due to the discovery of holoenzyme complexes containing Pol II, GTFs and other accessory proteins [32,33]. RHA is detected in Pol II holoenzyme complexes and interacts directly with Pol II [12,16,34]. In the present study, we have identified RHA as an actin-interacting protein in PICs. This conclusion is based on the following findings: (i) RHA and β-actin both exist in PICs; (ii) RHA associates with β-actin In vivo and in vitro; and (iii) disruption of the interaction between RHA and β-actin reduces the recruitment of Pol II into PICs. β-actin and RHA might serve as a molecular platform to recruit Pol II to the PIC, thus to participate in gene transcription regulation.
RHA is a nucleic acid helicase that unwinds both double-stranded DNA and RNA. Besides this, RHA has also been suggested to be a pre-RNA and mRNA-binding protein , which is involved in various steps of gene expression, including transcription, editing, splicing, RNA export and translation. Previously, two studies have revealed that there are functional links between actin and RHA in the nucleus. Zhang et al.  demonstrated that RHA mediated the attachment of nuclear ribonucleoprotein complexes to actin filaments, which may be related to RNA processing, transport, or other actin-dependent functions in the nucleus. These authors also suggested that an actin-based filamentous network may anchor RHA at the nucleolar periphery for pre-ribosomal RNA processing and ribosome assembly, and/or transport . In the present study, we show that RHA associates with β-actin in the nucleus (Figures 1B–1E), and an antibody against RHA reduces the recruitment of β-actin and Pol II into PICs (Figure 3A). We also reveal that both overexpression or depletion of RHA can affect β-actin-involved gene transcription regulation (Figures 5A–5C). These results suggest that the interaction between actin and RHA is involved in transcription. So it is possible that actin, in collaboration with RHA, exert functions throughout the entire gene expression process.
Functional domains of RHA include two copies of dsRBDs at its N-terminal known as dsRBD1 and dsRBD2. The conserved helicase domain is located in the central region and contains a DExH motif. The C-terminal contains a RGG-rich region that is characterized by preferential binding to single-strand nucleic acids. In the present study, we have identified two potential actin-binding sites by comparing RGG with the actin-binding motif of the myosin II head. In the following GST-pulldown assay, we have shown that the RGG region of RHA is responsible for the direct interaction with β-actin (Figure 2F). The actin monomer consists of four subdomains: subdomain 1 (amino acid residues 1–32, 70–144, and 338–375), subdomain 2 (amino acid residues 32–69), subdomain 3 (amino acid residues 145–180 and 270–337) and subdomain 4 (amino acid residues 181–269) . The actin–myosin head complex in the rigor state revealed several high-affinity sites on the actin molecule in subdomain 1 (amino acids 1–7, 18–28 and 40–113) [38,39]. In the present study, we demonstrate that the A1 mutant (amino acid residues 1–144) can bind to RHA (Figure 2B). Our results suggest that RHA interacts with β-actin directly through the potential sequences that are responsible for the actin–myosin complex.
Although β-actin is necessary for transcription by Pol II, there is no evidence to illustrate the direct interaction between β-actin and Pol II. In the present study, we show that the RGG region (amino acid residues 1150–1279) of RHA is capable of binding to β-actin. Because previous work has shown that the RHA fragment (amino acid residues 230–650) could interact with Pol II , it seems that RHA links β-actin with Pol II. The results of the present study demonstrate that the interaction between RHA and β-actin is important for the recruitment of Pol II into PICs (Figure 3B). Moreover, depletion of RHA interferes with the interaction of Pol II with β-actin (Figures 4D and 4E) and β-actin-involved gene transcription (Figures 5B and 5C). Taken together, we conclude that RHA acts as a bridging factor linking β-actin and Pol II, and functions in gene transcription regulation. In fact, RHA has also been reported to interact with several other nuclear proteins necessary for transcription activation, including CBP/p300, BRCA1, topoisomerase IIα , osterix  and the SMN (survival motor neuron) complex , so the bridging function may be general for the involvement of RHA in transcription.
This work was supported by the National Basic Research Program of China [grant number 2005CB5224004] and the National Natural Science Foundation of China [grant number 90608021].
We thank Jeffrey Parvin (Harvard Medical School, Boston, MA, U.S.A.) and Toshihiro Nakajima (Department of Genome Science, Institute of Medical Science, St. Marianna University School of Medicine, Japan) for providing plasmids of RHA, Wilma Hofmann (Department of Physiology and Biophysics, University of Illinois at Chicago, IL, Chicago, U.S.A.) for providing plasmid of AdMLP and helpful discussion.
Abbreviations: AdMLP, adenovirus major late promoter; BRCA1, breast-cancer susceptibility gene 1; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CSF-1, colony-stimulating factor-1; dsRBD, double-stranded RNA-binding domain; DTT, dithiothreitol; ECL, enhanced chemiluminescence; GST, glutathione transferase; GTF, general transcription factor; HA, haemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; IL, interleukin; IPTG, isopropyl β-D-thiogalactoside; MLE, maleless; Ni-NTA, Ni2+-nitrilotriacetate; PIC, pre-initiation complex; Pol II, RNA polymerase II; RGG, Arg-Gly-Gly; RHA, RNA helicase A; siRNA, small interfering RNA; TRITC, tetramethylrhodamine β-isothiocyanate
- © The Authors Journal compilation © 2009 Biochemical Society