Molecular determinants of nucleolar translocation of RNA helicase A

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Molecular determinants of nucleolar translocation of RNA helicase A Zhe Liu, Rachael Kenworthy, Christopher Green, Hengli Tang⁎ Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases

Received 29 March 2007

involved in multiple cellular processes and the life cycles of many viruses. The

Revised version received

subcellular localization of RHA is dynamic despite its steady-state concentration in the

11 July 2007

nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and

Accepted 27 July 2007

the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its

Available online 14 August 2007

carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to

Keywords:

nucleolus or the perinucleolar region. We found that low temperature treatment,

RNA helicase A

transcription inhibition or replication of hepatitis C virus caused the intranuclear

Nucleolus

redistribution of the protein, suggesting that RHA shuttles between the nucleolus and

Subcellular localization

nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon

ATPase

blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were

HCV

essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-

Transcription inhibition

stranded RNA binding domains (dsRBD II) was also required for this nucleolar

Cell survival

translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential

Nuclear localization signal

for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role. © 2007 Elsevier Inc. All rights reserved.

Introduction RNA helicase A (RHA), also named Nuclear DNA helicase II (NDH II), belongs to the DEAH-box family of nucleic acid helicases and is ubiquitously expressed in eukaryotic cells [1]. First isolated as an ATP-dependent RNA helicase [2,3], RHA was later shown to possess DNA-unwinding activity as well [4]. RHA has been implicated in numerous cellular functions, based mainly on its interaction with a variety of proteins and RNA. The subcellular localization of RHA is dynamic, shuttling between the cytoplasm and the nucleus despite its steady-state nuclear localization [5],

consistent with its potential functions in DNA transcription, RNA processing, translation and viral particle production [6–17]. The structural domain responsible for this shuttling activity has been mapped to the C-terminus of the protein and designated the nuclear transport domain (NTD) for its ability to direct both nuclear import and export of RHA [5]. The nuclear import signal within the NTD interacts with importin-α3 and requires arginine methylation to direct the nuclear localization of RHA [18,19]. The nuclear export mediated by NTD uses a yet to be identified pathway that is insensitive to leptomycin B, a drug that specifically blocks the CRM-1-dependent nuclear export pathway [5,20,21].

⁎ Corresponding author. Bio Unit I, Chieftan Way, Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA. Fax: +1 850 644 0481. E-mail address: [email protected] (H. Tang). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.07.037

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The localization of RHA is also dynamic within the nucleus. In human cells, RHA normally localizes in the nucleoplasm, excluded from the nucleolus in most cases [5,22,23]. However, a nucleolar accumulation of the protein was observed when these cells were treated with a transcription inhibitor or an agent that degrades F-actin [22,23], suggesting an association between RHA and the nucleolus. Consistent with this result, RHA was found to be among the 30 or so RNA helicases that are part of the nucleolar proteome [24]. In addition, immunostaining of endogenous murine RHA in several mouse cell lines revealed an unexpected enrichment of RHA in the nucleolus even when cells were not treated with any transcription inhibitors [25]. Together, these results suggest that nucleolar localization and translocation could prove relevant for the cellular functions of RHA. Like many other members of the helicase family, RHA has been implicated in the life cycles of viruses [12,17,26–29]. The dynamic subcellular localization of RHA is likely to be relevant for its participation in the replication of these viruses, especially those that either replicate in the cytoplasm or involve the nucleolus during replication [12,26,28]. Here we present evidence that RHA, a protein that binds to the 3′ UTR of hepatitis C virus (HCV) [29], alters its subnuclear localization in response to HCV replication as well as transcription inhibition. Instead of its usual nucleoplasmic localization, RHA accumulates in the nucleolus or the perinucleolar region in HCV replicon cells. The same subnuclear redistribution of RHA was observed when the cells were treated with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or cultured at 4 °C. Mapping experiments revealed that the NTD at the C-terminus is also important for nucleolar localization. Other important determinants include the ATPase motif and the second double-stranded RNA binding domain (dsRBD II), suggesting an involvement of RNA-binding in the nucleolar translocation of this protein.

Materials and methods Cells and treatments Huh-7.5 (a human hepatocarcinoma cell line), 293-FT (derived from human kidney fibroblasts), 3T3 (mouse embryos fibroblast cell), BHK-21 (derived from Syrian hamster kidney), HeLa (a human cervical cancer cell line) and COS-1 (derived from African green monkey kidney) cells were maintained in standard Dulbecco's minimal Eagle's medium (Hyclone, Logan, UT) supplemented with 10% of Fetal Bovine Serum (Cell Generation), 10 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma, St. Louis, MO). Clone B (HCV replicon) cells were maintained in the same media with the addition of 500 μg/ml of G418 (Cellgro, Antioch, IL). RNA polymerase II transcription was inhibited with 25 μg/ml 5,6-dichloro-1-β-Dribofuranosylbenzimidazole (DRB) (Sigma, St. Louis, MO) for 1.5 h for all experiments except the time-course study where the treatment times are indicated in the figure legend. Protein synthesis was inhibited by 50 μg/ml cycloheximide (Sigma) for 1.5 h. For the low temperature experiment, transfected cells were cultured at 4 °C for 1.5 h before fixation.

Molecular cloning and mutagenesis Plasmid pcDNA3.1(+)-myc-RHA was created with a multi-step cloning strategy. First, a 4.2 kb EcoRI fragment containing the fulllength cDNA of RHA was cut out of pcRHA [26] and cloned into the Eco I site of pcDNA3.1 (+) to make pcDNA3.1 (+)-RHA. PCR was then performed on the cDNA to create an myc-tag at the N-terminus. The forward primer (RHA5′-myc) introduces a BamHI site and myc-tag sequence upstream of the start codon of the RHA and it has a sequence of 5′-GCC GGA TCC ACC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG GGT GAC GTT AAA AAT TTT CTG-3′ (sequence complementary to RHA cDNA is underlined and the myc-tag sequence is italicized); the reverse primer (RHA 704-r) anneals to a sequence downstream of a unique SwaI restriction site and has the following sequence: 5′-GTT GTG ATC AGG ACC CAC-3′. The PCR product was digested with BamHI and SwaI and ligated to the similarly digested pcDNA3.1 (+)-RHA. This process adds an myc-tag fused to the N-terminus of the RHA and creates an myc-tagged full-length RHA expression cassette as a BamHI– EcoRI fragment. The K417R ATPase mutant was described previously [6,17] and similarly subcloned into the pcDNA3.1myc vector. Site-directed mutagenesis was performed to generate the S543L or DE510511AA mutation in pcDNA3.1 (+)-myc-RHA construct using a Quikchange™ kit (Stratagene, San Diego, CA). The mutagenesis primers have the following sequences: S543L-f: 5′-CCT GAA GTT CGC ATT GTT TTT ATG TTA GCT ACT ATT GAT ACC AGC ATG T-3′; S543L-r: 5′-ACA TGC TGG TAT CAA TAG TAG CTA ACA TAA AAA CAA TGC GAA CTT CAG G-3′; DE/AA-f: 5′-CGA GGA ATC AGT CAT GTA ATT GTA GCA GCA ATA CAT GAA AGA GAT ATT AAT ACT AG-3′; DE/AA-r: 5′-CTA GTA TTA ATA TCT CTT TCA TGT ATT GCT GCT ACA ATT ACA TGA CTG ATT CCT CG3′. Deletion mutant ΔRBD I was generated by digesting pcDNA3.1(+)-myc-RHA with BamHI and SwaI and ligating it to a 0.3 kb insert of the PCR product that removes the amino acids M1 through S130 and directly fuses the C-terminus of the myc-tag to residue G131 of RHA. The forward PCR primer (ΔRBDI-f) introduces the fusion and a BamHI site: 5′-GCC GGA GCC ACC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG GGC TAT GGT GTT CCT GGG C-3′ (sequence complementary to RHA cDNA is underlined and the myc-tag sequence is italicized); the reverse PCR primer, ΔRBDI-f, is the same RHA 704-r. ΔRBD II was produced by the removal of DNA fragment corresponding to amino acid M1 through P331 from pcDNA3.1(+)-myc-RHA as a BamHI and BstEII fragment and then the insertion of a 350 bp PCR product that encodes amino acids M1 through L117. The forward PCR primer was RHA5′-myc; the reverse PCR primer (ΔRBDII-r) anneals to the end of the dsRBD I and is followed by a BstEII site that allows the in-frame connection of residue L117 to reside W332 of RHA: 5′GCC GGT GAC CAA GAG GCC CCT ACC TCA GAA TT-3′ (sequence complementary to RHA cDNA is underlined). The double deletion mutant ΔRBDI+II was created in a similar manner except that only the myc-tag and the ATG start codon sequence was inserted back after the removal of the BamHI and BstEII fragment. The forward PCR primer (ΔRBDI+II-f) introduces a fusion between myc-tag and residue W332 and has the following sequence: 5′GCC GAA TCC ACC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG GAA CCA TCT CAG CGA CAA AAC-3′ (sequence complementary to RHA cDNA is underlined and the myc-tag sequence is italicized); the reverse PCR primer (ΔRBDI +II-r) anneals to a sequence downstream of the BstEII site on the RHA

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cDNA: 5′-CCA AGG ATT CCA GTT GGA TTG-3′. To make ΔNTDTNLS, pcDNA3.1(+)-myc-RHA was digested with BstXI and NotI to remove the C-terminus of the protein (amino acids Q1075 to the end) and then ligated to a 300 bp PCR product that encodes amino acids Q1075 to S1138 which was fused to a nuclear localization signal (NLS) of the SV40 large T antigen, followed by a stop codon. The forward PCR primer (ΔNTD-f) anneals to the upstream of the BstXI site in the cDNA and has the following sequence: 5′-CTT TGT ATT TGG TGA AAA G-3′; the reverse PCR primer (ΔNTD-r) introduces the fused NLS, a stop codon and the NotI restriction site: 5′-GCC GCG GCC GCC TAT ACC TTT CTC TTC TTT TTT GGA GAG ATC TGA CGG ATC ATG TTC-3′ (sequence complementary to RHA cDNA is underlined and the T-NLS sequence is italicized). The siRNA resistant version of the RHA cDNA was made by substituting the BamHI to SwaI fragment of the pcDNA3.1(+)-myc-RHA plasmid with a similar PCR product that introduces 8 silent mutations in recognition site of RHA-467 by way of the reverse PCR primer, which binds to a sequence just upstream of the SwaI site. The mutated siRNA site now has the sequence: 5′-GAG GTA CAG GCC ACA TTG GAG T-3′ instead of the wild-type sequence: 5′-GAA GTG CAA GCG ACT CTA GAA T-3′. The B23-GFP plasmid was gift from Dr. Robert Tsai.

Transfection

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RHA interference Lentiviral vector-based delivery and expression of shRNA have been described previously [30]. The target sequences for the siRNAs against RHA are as follows: RHA 42: 5′-AAG GAA GAT GAC CCC GCC TAT-3′; RHA 122: 5′-AAG GTT ATA ATT ACA CTG GCA -3′; RHA 467: 5′-AAG TGC AAG CGA CTC TAG AAT-3′; RHA 1136: 5′-AAG CAA TCT TGC AGG AGA GAG-3′. The primers for the sh-RHA 467 were 5′-GAA CTA GTG GAT CCG ACG CC-3′ and 5′-ggc GGA TCC AAA AAA gtg caa gcg act cta gaat TCT CTT GAA att cta gag tcg ctt gcac AAA CAA GGC TTT TCT CCA AGG G-3′ (the lowercase letters indicate the siRNA sequence). VSVG-pseudotyped lentivirus was packaged using the lentivirus support kit (Invitrogen). Huh-7 and replicon cells were transduced with standard methods.

Results Redistribution of RHA to the nucleolus and the perinucleolar region upon diverse cellular stress RHA has been reported to interact with a number of viruses [12,17,26–29]. In particular, it was identified as a member of a

Transfections of all the cell lines were performed with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA). Transfection media were replaced 6–16 h after transfection and cells were incubated for 48 h before being either fixed with 4% paraformeldehyde in 1× PBS for immunofluorescence staining or lysed with 1× lysis buffer (50 mM Tris–Cl, pH 8.0, 150 mM NaCl, and 1 mM PMSF) for western blotting.

SDS-PAGE and western blotting Cell lysates were fractionated on 10% SDS-PAGE and proteins were transferred to a ProtranTM nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a Trans-Blot apparatus (Bio-Rad, Hercules, CA). A mouse monoclonal anti-myc antibody (Sigma, St. Louis, MO; 1:3000 dilution) and goat-antimouse conjugated with HRP (Santa Cruz Biotechnology, 1:2000 dilution) were used to probe myc-tagged RHA proteins. The proteins were revealed by a western blot luminal reagent kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Immunofluorescence staining and confocal microscopy Cells grown on 15 mm coverslips in 12-well plates were fixed with 4% paraformaldehyde in PBS for 15 min and washed with 1× PBS and blocked with 1× PBTG (1× PBS, 2% of Triton X-100, 0.2% BSA, and 5% normal goat serum). Cells were incubated with anti-myc (1:500 dilution) for 2 h and washed for 30 min with 3 changes of washing buffer 1× PBT (1× PBS, 2% Triton X100), followed by staining with goat-anti-mouse antibody conjugated with FITC or TRITC (1:100 dilution) for 2 h. Finally, the nuclei were stained with DAPI. Fluorescence and DIC images of the cells were captured with a Zeiss LSM 510 Laser Scanning Confocal Microscope equipped with multi-photon laser and recorded with the accompanying LSMIB-3.2 software. Images were organized with Photoshop software.

Fig. 1 – RHA translocates to the nucleolar or perinucleolar area in HCV replicon cells. (A). Total protein in Huh-7.5 or Clone B cells lysates was separated by SDS-PAGE and subjected to immunoblotting with an anti-NS5A antibody to detect HCV protein expression in Clone B cells. Cyclophilin A, an abundant cellular protein, was used as a loading control. (B–D) Huh-7.5 cells (B) or Clone B cells (C–D) were transfected with myc-RHA which was detected by indirect immunofluorescence (green). RHA localized in the nucleoplasm in Huh-7.5 cells and displayed nucleolar exclusion (NoE) (B). RHA migrated to the perinucleolar region (C) or into the nucleoli (D) in Clone B cells. Scale bar = 10 μm. (E) Percentage of Huh-7.5 or Clone B cells exhibiting nucleolar translocation (NoT) (n N 100 cells for each cell line).

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Fig. 2 – RHA redistributes to the perinucleolar region or nucleoli in HeLa cells under DRB and low temperature treatment. (A–F) HeLa cells were transfected with myc-RHA for 48 h and then subjected to the various treatments indicated for 1.5 h before being fixed for staining with anti-myc (green) and DAPI (blue). 25 μg/ml DRB and 50 μg/ml CHX were used throughout the study. RHA localized in the nucleoplasm in HeLa cells in the absence of treatment (A). RHA translocated to nucleoli (B–E) or the perinucleolar region (C–F) in HeLa cells treated with DRB or low temperature, respectively. CHX treatment did not result in the nucleolar translocation of RHA (D). Scale bar = 10 μm. (G) HeLa cells were co-transfected with myc-RHA and GFP-B23 and similarly treated with DRB before being stained with anti-myc (red) and DAPI (blue). Scale bar = 10 μm.

group of proteins that interacts with the genomes of two related RNA viruses: bovine viral diarrhea virus (BVDV) and hepatitis C virus [28,29]. RNA interference demonstrated an essential role of RHA in the replication of BVDV [28]. Since both BVDV and HCV replicate in the cytoplasm and we previously found that RHA shuttles between the nucleus and the cytoplasm [5], we were interested in studying if HCV replication has any effect on the subcellular localization of the protein, more specifically, if RHA would re-locate to the cytoplasm in HCV replicon cells. We transfected an myc-tagged form of RHA into a pair of human hepatoma cells with or without a replicating HCV replicon (Fig. 1A, [31,32]) and then examined the subcellular localization of RHA by indirect immunofluorescence staining. The steady-state localization of the myctagged RHA in the majority (N95%) of the transfected Huh-7.5 (naive) cells was exclusively nucleoplasmic and excluded from the nucleoli (Figs. 1B, E). This result mirrors that of endogenous RHA in human cells reported previously [5,22,23]; here we designate this distribution of RHA as nucleolus excluded (NoE). In contrast, a dramatically different staining pattern was observed for myc-RHA in the Clone B cells where HCV RNA actively replicates. The fluorescence was enriched in either the perinucleolar region or the nucleolus over 95% of the transfected cells, with a concurrent decrease in staining in the nucleoplasm (Figs. 1C, D). We designated this localization pattern as nucleolus translocated (NoT). Although HCV replication

did not cause RHA to translocate to the cytoplasm, which is the site of viral replication, this result did indicate that HCV replication has the capacity to alter the steady-state localization of RHA.

Fig. 3 – Time-course of RHA's nucleolar translocation in HeLa cells in the presence of DRB. HeLa cells were transfected with myc-RHA and treated with DRB for 0, 5, 15, 30, 45, 60, 75 or 90 min before fixation and staining. Percentage of cells showing migration of RHA from nuclei to nucleoli or perinucleolar region was calculated at various time points (n N 100 cells for each time points) post addition of the drug.

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To examine if this phenomenon is specific to viral replication or can be induced by general cell stress, we tested a number of other stress-inducing conditions including 4 °C incubation, transcription inhibition by 5,6-dichloro-1-β-Dribofuranosylbenzidazole (DRB) and translation inhibition by cycloheximide (CHX). We performed these experiments

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in HeLa cells. Both low temperature and DRB treatment effectively caused nucleolar (Figs. 2B, E) and perinucleolar translocation (Figs. 2C, F) of RHA. Translation inhibition with cycloheximide did not cause NoT of RHA (Fig. 2D). The perinucleolar nature of the ring-like staining for RHA was confirmed by co-transfecting a nucleophosmin/B23-GFP

Fig. 4 – The NTD and ATPase motifs of RHA are essential for nucleolar and perinucleolar translocation. (A) The RHA protein also domain structure with the locations of the ATPase mutants designated as arrows; mutants with the deletion of dsRBDs are shown. (B) Western blotting results demonstrating normal expression and PAGE migration patterns of the mutants. (C, D) Localization of RHA ΔNTD-TNLS in the absence or presence of DRB treatment. (E–J) Localization of ATPase mutants ( K417R, the S543L or DE510/511 AA) in the absence or presence of DRB treatment. None of ATPase mutant RHA was able to migrate to nucleolar or perinucleolar region in the presence of DRB. Scale bar = 10 μm. (K–M). The ATPase mutant K417R failed to translocate to the nucleolus or the perinucleolar region in HCV replicon cells. Clone B cells were transfected with either wild-type (K, L) or the ATPase mutant RHA K417R (M) and stained with anti-myc (green) 48 h post-transfection. Scale bar = 10 μm.

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Table 1 – Subcellular localization of deletion mutants of RHA RHA

Full-length ΔdsRBD I ΔdsRBD II ΔdsRBD I + II ΔNTD-TNLS

Subcellular localization for over 90% the transfected cells (−) DRB

(+) DRB

NoE NoE or NoT NoE NoE NoE

NoT NoE or NoT NoE NoE NoE

construct to mark the nucleoli with green fluorescence while detecting the myc-tagged RHA with a Rhodamine-conjugated secondary antibody (Fig. 2G). We next examined the time-course of the redistribution of RHA upon DRB treatment. We treated the myc-RHA transfected cells for various amounts of time with 25 μg/ml DRB and then fixed the cells for staining. Without DRB treatment, a small percentage (5–6%) of the transfected cells exhibited NoT while the remainder exhibited NoE phenotype. As early as 5 min after addition of the drug, nucleolar or perinucleolar accumulation of RHA was observed in over 20% of the transfected cells (Fig. 3). The percentage of cells with the NoT phenotype steadily increased over time and

reached a maximum (∼ 95%) at approximately 1.5 h posttreatment.

The nuclear transport domain of RHA is important for its nucleolar localization RHA harbors a nuclear transport domain (NTD) at its Cterminus, overlapping the RGG domain [5]. NTD contains sequences that specify both the nuclear import and export function of the protein [5,18,19]. We were interested in any role that NTD may play in the nucleolar localization of RHA. Deletion of NTD from the full-length RNA resulted in a protein that localized exclusively to the cytoplasm ([5], data not shown), precluding the examination of any role of this domain in nucleolar translocation. To direct the NTD-deleted RHA into the nucleus, we replaced the NTD with a classical NLS from the SV40 large T antigen (Fig. 4A). The subcellular localization of this chimerical protein, termed ΔNTD-TNLS, was examined along with that of the wild-type RHA in HeLa cells. When cells were not treated with drug, the chimerical protein was located to the nucleoplasm and was excluded from the nucleolus, much like the wild-type protein (Fig. 4C). This NoE phenotype was maintained for ΔNTD-TNLS in DRB-treated cells (Fig. 4D), in contrast to the situation with the wild-type protein (Fig. 2). This result suggests that the NTD domain of RHA is required for its nucleolar translocation and the classical NLS of the

Fig. 5 – RHA displays a similar NoT pattern in COS-1, BHK and 3T3 cells upon DRB treatment. COS-1 (A–C), BHK (D–F) and 3T3 cells (G–I) were transfected with myc-RHA for 48 h, followed by immunostaining with (B, C, E, F, H, I) or without (A, D, G) DRB treatment. RHA exhibited NoT in the presence of DRB in all three cell lines. Scale bar = 10 μm. (J) Percentages of COS-1, BHK or 3T3 cells displaying nucleolar translocation (n N 100 cells for each cell line).

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SV40 large T antigen, a viral DNA helicase, does not contain the signal for nucleolar targeting. It also ruled out the possibility that the NoT of RHA was a result of “leaky” nucleoli caused by stress.

Mutations abolishing the ATPase activity of RHA prevent the nucleolar translocation Next, we investigated if the ATPase activity of RHA is needed for its nucleolar translocation; we generated several mutant cDNAs of RHA in which the critical ATPase motifs have been mutated, including a previously reported helicase mutant K417R [7] and two new mutants S543L and D510A/E511A. K417 and S543 are key residues involved in ATP binding and D510 and E511 are part of the helicase signature motif of the DEAHbox helicase family to which RHA belongs. We expressed these mutant proteins in HeLa cells and examined their subcellular localization. Without DRB treatment, all proteins exhibited the NoE phenotype (Figs. 4E, G, I). When the cells were treated with DRB, none of the mutant proteins was able to translocate to the nucleolus or the perinucleolar region, in sharp contrast to the situation with the wild-type protein (Figs. 4F, H, J). Similar results were obtained with other types of stresses; the ATPase deficient mutant K417R failed to translocate to the perinucleolar region in HCV replicon cells (Fig. 4M) or in cells incubated at 4 °C (data not shown). The electrophoresis mobility and stability of the protein in transfected cells were not affected by the mutations (Fig. 4B). This result suggests that

Fig. 7 – NTD contains a signal for perinucleolar targeting. Expression plasmids for myc-tagged Tap-X and Tap-X-NTD have been described previously [37]. HeLa cells were transfected with Tap-X (A, B) or Tap-X-NTD (C, D) for 48 h followed with or without DRB treatment. Both Tap-X and Tap-X-NTD localized in the nucleoplasm in the absence of DRB (A, C). In the presence of DRB, the subnuclear localization of Tap-X did not change (B) while Tap-X-NTD showed concentration in the perinucleolar region in over half of the transfected cells (D). (E, F) Enlarged views of the bottom cell in (D), arrows indicate accumulation of Tap-X-NTD around the nucleoli.

the mechanism of RHA's nucleolar translocation requires the ATPase activity of the protein.

The second dsRNA-binding domain of RHA is involved in the nucleolar translocation of the protein RHA contains two double-stranded RNA-binding domains (dsRBD I and II) at the N-terminus [33]. We next examined the importance of the RNA-binding ability of RHA for NoT. Deletion mutations were made for each of these domains as well as a double deletion mutant in which both dsRBD I and dsRBD II were deleted. We then transfected HeLa cells with these mutants and examined their ability to translocate to the nucleolus upon DRB treatment. The results are summarized in Table 1. While dsRBD I is dispensable for NoT, dsRBD II is absolutely essential as either the double deletion of dsRBD I and II or the single dsRBD II deletion completely abolished the ability of RHA to translocate to the nucleolus upon DRB treatment.

The nucleolar translocation of RHA is not restricted to human cells Fig. 6 – Subcellular localization of two other RNA-binding proteins. HeLa cells were transfected with GFP-hnRNPA1 (A, B) or GFP-SRp30c (C, E) for 48 h followed with or without DRB treatment. Both hnRNPA1 and SRp30c localized in the nucleoplasm in the absence of DRB (A, C). In the presence of DRB, the subnuclear localization of GFP-hnRNPA1 did not change (B) while GFP-SRp30c redistributed to the perinucleolar or nucleolar area (D, E). Scale bar = 10 μm.

The subcellular localization of the murine homologue of RHA has been shown to be different from the human protein and concentrated in the mouse nucleoli [25]. It was not clear whether this difference in subcellular localization was due to inherent differences between the murine and human proteins themselves or determined by the cell type. Here we addressed this question by expressing the human RHA cDNA in cells of several different species. We tested COS-1 (African Green

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Monkey kidney), BHK (hamster kidney) and 3T3 (mouse fibroblast) cell lines. In all three cell types, the myc-tagged human RHA exhibited NoE localization in the majority of the cells, consistent with endogenous human protein but different from endogenous mouse RHA (Figs. 5A, G, D). In addition, treatment with DRB drastically increased percentage of the cells with the NoT phenotype in all the three cell lines. Both the perinucleolar and nucleolar translocations were observed in all these cells, indicating that the nucleolar translocation mechanism used by human RHA is conserved in these cells. In all cases, NoT was observed in a small percentage of cells (∼2% for COS-1 and BHK; ∼ 6% in 3T3 cells) without any drug treatment while treatment dramatically increased this percentage (Fig. 5J). This spontaneous redistribution of RHA suggests a natural association of RHA with the nucleolus [24] in a variety of species.

A RNA-binding protein exhibits NoT phenotype upon transcription inhibition To further study the generality of nucleolar translocation of RNA-binding proteins, we examined the subcellular localization of two RNA-binding proteins that also exhibit steadystate NoE. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multi-functional RNA binding protein that

shuttles between the nucleus and the cytoplasm [34]. We expressed a GFP-hnRNP A1 fusion protein in HeLa cells and treated the cells with DRB. The protein localized in the nucleoplasm and did not redistribute to the nucleolus with DRB treatment for up to 1.5 h (Figs. 6A, B). The second RNAbinding protein we studied was SRp30c, a splicing factor that was identified as a RHA-binding protein in a yeast two-hybrid screen ([35], Reddy TR and Tang H, unpublished data). GFPSRp30c exhibits a NoE phenotype in transfected HeLa cells (Fig. 6C), and DRB treatment caused a NoT localization pattern that is very similar to that of RHA under the same treatment (Figs. 6D, E). These results demonstrate that nucleolar redistribution is neither a general feature of the RNA-binding proteins nor specific to RHA.

NTD contains a signal for perinucleolar targeting We next examined the ability of NTD to target another RNAbinding protein to the nucleolus. Human Tap is a RNAbinding protein that shuttles between the nucleus and the cytoplasm, similar to RHA [36]. We have previously constructed a C-terminal truncated Tap protein, Tap-X, which localized to the nucleus [37] but did not translocate to the nucleolus upon DRB treatment (Figs. 7A, B). A hybrid protein (Tap-X-NTD) with the NTD in the place of the authentic Tap

Fig. 8 – Suppressing RHA expression using RNA interference induces apoptosis of human hepatoma cells in vitro. (A) Inhibition of RHA protein expression by siRNAs targeting its mRNA. siRNA duplexes were transfected into Huh-7 cells and total proteins were extracted from the transfected cells 4 days post-transfection and then subjected to SDS-PAGE and immunoblotting. The numbers in the names of the siRNAs indicate the nucleotide position of the siRNA target in RHA mRNA. (B) Puromycin-selectable lentiviral vectors expressing either a control siRNA targeting firefly luciferase or si-RHA 467 were co-transfected into Huh-7 cells along with various cDNAs of RHA as indicated: WT: wild-type; K417R: ATPase mutant; ΔNTD: ΔNTD-TNLS. All cDNAs were in the siRNA resistant form. The double transfected cells were then subjected to puromycin selection for 2 weeks. (C–H) Lentiviral vectors expressing either GFP alone or in combination with the si-RHA 467 were introduced into Huh-7 cells via transduction. The cells were fixed for immunofluorescence analysis 96 h post-transduction. The nuclei were stained with DAPI and transduced cells were identified by the expression of GFP.

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C-terminal domain [37] was also distributed uniformly in the nucleoplasm in the absence of treatment (Fig. 7C). Upon DRB treatment, however, the localization pattern of Tap-X-NTD changed from the even distribution in the nucleoplasm to noticeable accumulation in the perinucleolar regions (Figs. 7D–F). This perinucleolar targeting was observed in over half of the cells transfected with Tap-X-NTD, but not in any of the Tap-X transfected cells. Interestingly, the site of accumulation appears to overlap with areas of less intense staining of DNA (Figs. 7E, F), which was also observed for wild-type RHA (data not shown). Together, these results suggest that NTD contains a targeting sequence for the perinucleolar region.

RNA interference of RHA expression results in apoptosis of hepatoma cells in vitro To investigate the function of RHA, we designed small interfering RNAs (siRNA) to suppress its expression. Four siRNAs targeting various sites of the RHA mRNA were constructed and tested. Control siRNAs included siRNAs targeting firefly luciferase or GAPDH. One of the siRNAs, si-RHA 467, was the most potent in knocking down RHA expression at both the RNA (data not shown) and the protein level (Fig. 8A). We then cloned si-RHA 467 as a small hairpin RNA (shRNA) in a lentiviral vector with a puromycin-selectable marker [30] and introduced the vector into Clone B cells. Repeated attempts to select stable cells expressing the shRNA were not successful while we could easily obtain stable cell lines expressing shRNAs against a variety of other genes. This suggested to us that RHA is essential for the survival of the Huh-7 cells, on which the replicon cells were based. To unequivocally prove this, we performed rescue experiments with a RHA cDNA that contains silent mutations in the siRNA recognition site. When a cDNA encoding wild-type amino acid sequence of RHA was co-transfected with the si-RHA 467, a rescue of cell survival was observed (Fig. 8B). A cDNA bearing the K417R mutation was less efficient in the rescuing function, indicating the involvement of the ATPase motif in RHA's essential role in cell survival. Surprisingly, the RHA mutant that lost its ability to translocate into the nucleolus (ΔNTD-TNLS) still rescued cell survival in this assay with an efficiency equivalent to the wildtype protein (Fig. 8B), suggesting that nucleolar targeting is not important for RHA's function to maintain cell viability in vitro. To further study the mechanism of si-RHA 467 mediated cell killing, we transferred the shRNA cassette into an HIV-based vector containing a GFP gene as the selectable marker [30]. The shRNA vector was then delivered into Huh-7 cells along with an empty vector expressing GFP alone (without the shRNA) as a control. In the cells transduced with the control vector, the DAPI staining and the morphology of the nuclei looked normal in both GFP-positive and GFP-negative cells (Figs. 8C, D). In contrast, when the shRNA vector was introduced, extensive abnormality of the nucleus was observed in almost all the GFP-positive cells while the GFP-negative cells remained normal (Figs. 8E–H). Nuclear blebbing and formation of apoptotic bodies, both classic signs of cells undergoing apoptosis, were observed. Eventually all the GFP-positive cells died, mirroring the results in the puromycin selection experiment. The same results were obtained with Clone B cells with an HCV replicon (data not shown).

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Discussion Nucleolar localization and transport have been reported for other DEAD-box proteins. An3 protein, an RNA helicase expressed in Xenopus laevis oocytes, is a nucleo-cytoplasmic shuttling protein that associates with the nucleolus at certain stages of development [38,39]. A similar development stagedependent nucleolar association was also observed for RHA in bovine oocytes [40]. Another DEAD-box helicase, p68, has been shown to redistribute to the nucleolus in a cell cycledependent manner and interact with fibrillarin, a resident nucleolar protein [41–43]. In both yeast and human cells, the splicing factor Prp43p, which is a putative DEAD-box RHA helicase (DDX-15), is detected in the nucleolus [44,45]. DDX47 and DDX18, two putative RHA helicases, were identified to interact with the nucleolar protein NOP132 and localize to the nucleolus [46]. Interestingly, for several of these proteins, transcriptional inhibition altered the subnuclear localization of the helicase protein, redistributing them from the nucleolus to the nucleoplasm. RHA has been identified as part of the nucleolar proteome using a biochemical approach [24]. In that study, the association of RHA with the nucleolus was enhanced when cells were treated with the transcription inhibitor actinomycin D, supporting results obtained using immunofluorescence staining [22]. We showed here that there are two possible localization patterns of RHA after DRB treatment: nucleolar and perinucleolar. The relative percentage of these two types of subnuclear localizations varied between experiments as well as cell lines and they may represent two distinct “stop points” in the transport pathway of the protein cycling between the nucleolus and nucleoplasm. At least two hypotheses could be explored to explain the observed nucleolar redistribution of RHA upon transcription inhibition. In the first model, the nucleolar and perinucleolar localization of RHA is an abnormality caused by cellular stresses imposed either by viral replication or transcription inhibition. In the second model, RHA is an intranuclear shuttling protein that rapidly cycles between the nucleolus and nucleoplasm. Its steadystate concentration in the nucleoplasm could be a result of either the different rates of transport to and from nucleolus or the time of retention in the different subnuclear compartments. If the movement of RHA from nucleolus to nucleoplasm is selectively blocked, then the protein would accumulate in the nucleolus. This later hypothesis is supported by two lines of evidence: first, RHA is normally found in the purified nucleolus; second, incubating cells at 4 °C resulted in NoT of RHA, consistent with the notion that the blockade of an energy-dependent transport pathway is responsible for the redistribution phenotype. It has been difficult to ascertain the contribution of nucleolar localization to the cellular function of these bona fide or putative RNA helicases. The frequent observation that transcription inhibition can regulate nucleolar translocation suggests a link between a role in ribosomal RNA biogenesis by these RNA helicases and their association with the nucleolus. In fact, endogenous murine RHA has been reported to localize to nucleolus in the absence of drug treatment [25]. Our results here now show that human RHA, when expressed in mouse

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cells, does not have the same nucleolar enrichment under the same conditions. This suggests that sequence difference between murine and human RHA protein accounts for the distinct subcellular localization patterns. Interestingly, NTD is the region with the most divergence between murine and human RHA in the amino acid sequence. The homology is only ∼ 30% compared to the overall ∼90% homology between the proteins from the two species. Moreover, the RGG box of the murine RHA cDNA, which is immediately downstream of the NTD, is significantly extended compared to the human counterpart [25]. Unlike the nuclear localization signal (NLS) and the leucinerich nuclear export signal (LR-NES), the signal of targeting proteins to the nucleolus is much less defined. So far, no consensus sequence has been recognized for nucleolar targeting although there seems to be an enrichment of positively charged residues in the nucleolar localization sequences (NoLS) identified so far [47]. Here we show that the NTD of RHA, which contains both an NLS and an atypical NES, also harbors the sequence necessary for directing the protein into the nucleolus. When NTD is deleted from RHA and replaced with a classical NLS from a viral DNA helicase, the chimerical protein was not able to translocate into the nucleolus or to the perinucleolar region upon transcription inhibition. However, it did bring the protein into the nucleus as expected, indicating that the NLS and NoLS of RHA are distinct properties. In principle, a protein could also localize to the nucleolus by virtue of an interaction between the protein and components of the nucleolus. In other words, other than the NLS for nuclear import, further targeting of the protein to the nucleolus may not require a discrete signal but rather be dependent upon specific domains involved in the interaction with resident nucleolar proteins or RNA. In agreement with this hypothesis, it has been demonstrated that RNA-binding domains are usually important for the nucleolar translocation of various RNA-binding proteins [48,49]. Deletion of the first dsRBD of RHA did not prevent nucleolar translocation of the protein but resulted in a high percentage (∼50%) of the cells with the NoT phenotype without any drug treatment, indicating that dsRBD I may contribute to the nucleolar-nucleoplasmic shuttling ability of the protein. Deletion of dsRBD II alone as well as both dsRBDs abolished RHA's ability to relocate to the nucleolus, demonstrating an important role for this domain in the NoT phenotype. In addition, although fusion of NTD to the Cterminus of an mRNA exporter receptor, Tap, resulted in a certain degree of perinucleolar targeting, we did not observe complete nucleolar translocation for this hybrid protein, suggesting that specific interactions between RNA-binding and nucleolar targeting signal may be required for efficient translocation. The regulation and energy requirement of nucleolar translocation are just beginning to be understood. A GTPdriven cycle has been shown to be involved in determining the nucleolar residency of nucleostemin [50]; raising the possibility that protein cycling between the nucleolus and nucleoplasm could be regulated by signaling pathways involving G proteins. Many intracellular transport processes are accomplished by a combination of passive diffusions and energydependent active transport. Our finding that ATPase motifs are important for the nucleolar localization of RHA suggests that

the translocation process is ATP-dependent. A similar ATP hydrolysis-dependent nucleolar translocation of tumor suppressor protein p53 has also been recently described [51]. The interaction between viruses and the nucleolus has been documented for many different viruses including DNA virus, retroviruses and RNA viruses. A role of the nucleolus in the replication of these viruses is highlighted by the many examples of interaction between viral proteins and nucleolar antigens [52]. Even more viral proteins could localize to the nucleolus under various conditions, further supporting the view that the nucleolus could affect and/or be affected by viral replication. It is currently unclear what role, if any, that RHA's nucleolar localization has in the replication of the various viruses with which it interacts [12,17,26–29]. In the case of HCV, although RHA has been identified as a cellular protein associated with HCV 3′UTR in replicon cells and HCV replication is shown here to alter the subcellular localization of RHA, definitive functional evidence of RHA's involvement in HCV replication is lacking. On the other hand, gene profiling showed only subtle changes in gene expression in HCV replicon cells [53], making global altercations of mRNA and protein subcellular localizations as a result of HCV replication unlikely, which argues for a fairly specific interaction between HCV replication and the RHA NoT pathway. RNA interference experiments showed that RHA is essential for the survival of human hepatoma cells in vitro. Suppressing RHA expression induced classic signs of apoptosis and eventually led to cell death. Wild-type and NTD-deleted RHA rescued the lethal phenotype of RNAi while a ATPase mutant was much less efficient in doing so, indicating an involvement of the ATPase/helicase, but not the nucleolar targeting, activity in this function. Consistent with our results, previous studies identified RHA to be a target of caspase-3 and the cleavage of RHA by caspase may be an important part of the process leading to programmed cell death [54,55]. In mouse models, homozygous loss of murine RHA was embryonic lethal [56]. In summary, we showed here for the first time that the HCV replication could alter the subcellular localization of a human DEAH-box RNA helicase and cause it to associate with the nucleolus. This redistribution of RHA in HCV replicon cells was indistinguishable from the change caused by the transcription inhibitor DRB or low temperature treatment, indicating a common pathway for translocation of RHA to the nucleolus and perinucleolar regions. The domains involved in this translocation are the C-terminal NTD, ATPase motifs and the dsRBDII. The ATPase motif contributes to RHA's function in sustaining cell viability while the NTD is dispensable for this role.

Acknowledgments This work was supported by American Heart Association National Center. We thank Dr. Charlie Rice (Rockefeller University) for providing Clone B cells, Dr. Robert Tsai for B23-GFP plasmid and Dr. C-G Lee for the original RHA cDNA clone and anti-RHA sera. Dr. Kimberly Riddle helped with confocal imaging and the molecular cloning core facility of FSU biology department is acknowledged for assisting us with mutagenesis and plasmid construction. We would also like to thank Diana Lambert for proof-reading the manuscript.

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