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¹ Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan
² Department of Genetic Diagnosis, Gifu International Institute of Biotechnology, 1-1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, Japan
³ Department of Anatomy, Nagoya University, School of Medicine, Tsurumai, Showa-ku, Nagoya 466-8550, Japan

	Abstract

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Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells.

	Introduction

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Structural changes in RNA biologically play an important role in almost every cellular process within living organisms and are attributed to the activity of RNA helicases (Tanner & Linder 2001). Such processes include transcription, translation, ribosome biogenesis, mRNA splicing, RNA chaperones, RNA maturation and RNA degradation. DEAD-box RNA helicases, named after the conserved Asp-Glu-Ala-Asp (DEAD) amino acid sequence, are members of helicase Superfamily II (SFII) (Gorbalenya et al. 1989; Linder et al. 1989). DEAD-box proteins are thought to possess both RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, and to be responsible for duplex RNA unwinding, which facilitates the rearrangement of the RNA structure. Sequence analysis, as well as biochemical and genetic experiments, has revealed that helicases are composed of two ‘core’ domains with highly conserved motifs (Gorbalenya et al. 1989; Pause et al. 1993; Tanner 2003). Motif I (otherwise known as the Walker A motif), which consists of the consensus sequence G/AxxxxGKT/S, comprises the phosphate binding loop, or the so-called P-loop, forming a pocket that binds the ß- and -phosphates of ATP. The open or closed conformations of the P-loop are adjusted by interactions involving the residues around it. The open conformation, which sterically complements the phosphates, is generally found in ATP binding proteins. The closed conformation has never been seen in any other crystal structures of NTPases, including RNA or DNA helicases, with or without ATP. The closed P-loop fills the ATP binding pocket and sterically inhibits ATP binding. The Q-motif, a newly identified motif upstream of motif I, is believed to be involved in regulation of ATP binding and hydrolysis by modulating the state of the P-loop (Tanner 2003; Cordin et al. 2004). Such a motif is unique to the DEAD-box RNA helicase family. The GG motif, another conserved sequence in DEAD-box proteins, is also unique to this family. It should be noted that mutating the glycine residues of the GG motif in yeast eukaryotic initiation factor 4 A (eIF4A) to aspartic acid leads to a lethal phenotype and severe growth defects (Schmid & Linder 1991). Although the GV sequence of the Hepatitis C virus (HCV) NS3 helicase (Kim et al. 1998), which is equivalent to the GG motif, contacts nucleic acids, it is believed that the same region in eIF4A is also involved in binding to other factors.

Human rck/p54, a product of a lymphoma-linked chromosomal translocation breakpoint gene on 11q23 (Akao et al. 1992; Lu & Yunis 1992), is a member of the DEAD-box RNA helicase family, and consists of 472 amino acids with a molecular weight 53.2 kDa. In addition, rck/p54 is the prototype of the RCK subfamily in DEAD-box RNA helicase family (Minshall et al. 2001). eIF4A, which is considered to be a minimal DEAD-box protein, has often been studied as the prototype of this family. On the contrary, efforts to elucidate the functions of other members of the RCK subfamily with unique extensions of 80 residues at the N-terminus has been poor (Fig. 2). Although expression of rck/p54, which is ubiquitously present, is very poor in brain, skeletal muscle and lung tissues, a significant amount of rck/p54 was detected in tumors originating from these tissues (Akao et al. 1995). In some colorectal adenomas, which are premalignant lesions of colon cancer, rck/p54 and c-myc were found to be co-over-expressed (Nakagawa et al. 1999; Hashimoto et al. 2001). Electron microscopic analysis demonstrated that rck/p54 binds to c-myc mRNA and exhibits RNA unwinding activity toward c-myc mRNA in vitro (Akao et al. 2003). These previous reports further implied that rck/p54 contributes to cell proliferation and/or carcinogenesis at the translation level or the RNA processing step.

View larger version (96K): [in this window] [in a new window]   Figure 2  Secondary structure elements of Nc-rck/p54 and sequence alignments of 11 members of the DEAD-box RNA helicase containing rck/p54. Sequences were aligned with the program CLUSTAL_W (Thompson et al. 1994). Secondary structure elements of Nc-rck/p54 (70–288) are described using cylinders ( helices) and arrows (ß strands). Comparison is made with the previously determined crystal structures of three proteins: yeast eIF4A, Methanococcus janaschii MjDEAD and Bacillus stearothermophilus BstDEAD and seven members of RCK subfamily: mouse rck/p54 (Accession Number: D50494), X. laevis Xp54 (X92421), S. cerevisiae Dhh1 (X66057), S. solidissima Clamp47 (AF399934), C. elegans Cgh-1 (AC006605), S. pombe Ste13 (D29795) and D. melanogaster ME31B (M59926). Conserved motifs of N-terminal domain is shown as black background.

In the DEAD-box family, only three crystal structures have been reported; the N- and C-terminal domains of yeast eIF4A (Benz et al. 1999; Johnson & McKay 1999; Caruthers et al. 2000), MjDEAD from Methanococcus janaschii (Story et al. 2001) and the N-terminal domain of BstDEAD from Bacillus stearothermophilus (Carmel & Matthews 2004). Based on those structures, the inchworm model, which is a well-known model for monomer helicases, has been advocated as the reaction mechanism for DEAD-box proteins (Tanner & Linder 2001). However, for most of these proteins, the precise mechanisms and dynamics of substrate recognition remain unknown.

In the present paper, we provide structural and biological evidences to reveal the dynamics of substrate recognition of rck/p54, which can further provide evidence for the inchworm model.

	Results

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Overall structure of Nc-rck/p54

Two Nc-rck/p54 molecules, chains A and B, were identified; a tartaric ion was found to bind only with chain B and one Zn²⁺ ion was present in the asymmetric unit. No electron density was seen for residues 70-82 on either chain. There were few significant differences between the two chain structures, the r.m.s. deviation between chains A and B being 0.55 Å for the C atoms. The region bearing the GG motifs in chain A is relatively disordered in comparison with that in chain B. For this reason, we will hereafter discuss chain B. Nc-rck/p54 has a parallel /ß structure having a fold with RecA-like topology that is similar to corresponding domains in related helicases (Fig. 1A). Superpositions of Nc-rck/p54 on the N-terminal domains of yeast eIF4A, Methanococcus janaschii MjDEAD and Bacillus stearothermophilus BstDEAD yield r.m.s. deviations of 0.91 Å, 1.10 Å and 0.98 Å for 190, 193 and 183 equivalent C atoms, respectively, suggesting their structural similarity. Furthermore, this similarity also extends to more distantly related proteins in Superfamily I (SFI) and Superfamily II (SFII). Superpositions of the RecA-like core of Nc-rck/p54 on that of NS3 helicase from HCV, a member of the DexH-box family of SFII, and on that of PcrA from Bacillus stearothermophilus, an SFI DNA helicase, demonstrate the conservation of the RecA-like core among various helicase families (r.m.s. deviations of 1.72 Å and 1.51 Å for 83 and 99 C atoms, respectively).

View larger version (39K): [in this window] [in a new window]   Figure 1  Structure of Nc-rck/p54. (A) Ribbon representation of Nc-rck/p54 B chain consisting of residues 83-288. The color-coding used to distinguish the conserved motifs is maintained through the other figures as follows: Q-motif, purple; motif I (P-loop or Walker A), blue; motif Ia, light blue; motif Ib, pink; motif II (Walker B or DEAD), red; and motif III (SAT), yellow. A bound tartaric ion is shown as a stick model. (B) Comparison of P-loop conformations with reported helicases. The P-loops of Nc-rck/p54 (blue, PDB code; 1vec), BstDEAD (Orange, PDB code;), MjDEAD (green, PDB code; 1hv8) and eIF4A (brown, PDB code; 1qva) are seen in the closed conformations in comparison with open conformations of eIF4A (pink, PDB code; 1qd), PcrA (purple, PDB code; 3pjr) with ATP (cyan). The P-loop of Nc-rck/p54 forms the most closed conformation among the reported structures. Motif II and "DEAD," and III and "SAT," are shown in red and yellow, respectively, as is also the case in C and D. (C) Interactions between residues in the closed conformation of Nc-rck/p54. Distances between residues are shown as dashed lines and are given in Å, as is also the case in (D). (D) Interactions between Asn 238 in motif II and residues in motif III. (E) Comparison of ATP binding site of Nc-rck/p54 (red), including Q motif, with open P-loop conformation of eIF4A (blue).

The P-loop of Nc-rck/p54 forms a closed conformation (Fig. 1B), similar to that described in the structure of eIF4A (Johnson & McKay 1999), MjDEAD (Story et al. 2001), BstDEAD (Carmel & Matthews 2004), and PcrA (Velankar et al. 1999). This closed conformation in Nc-rck/p54 was the result of interactions between motifs, as described below.

The Asn131 side-chain in the P-loop is involved in water-mediated binding to Asp238 in motif II (the DEAD motif), and to Ala267 in motif III (the SAT motif) (Fig. 1C). In the DEAD-box RNA helicase family, 94% of residues at position 131 in rck/p54 is Ser or Thr; however, the substitutions of Ser or Thr to Asn in motif I frequently appears in proteins belonging to the RCK subfamily (Fig. 2) (Ladomery et al. 1997; Minshall et al. 2001; Tanner 2003). In eIF4A and BstDEAD, the residues corresponding to Asn131 in Nc-rck/p54 are Ser67 and Thr50, respectively. Interestingly, single-point mutants of Asn131 to Ser or Thr in rck/p54 are hardly expressed, even if the method of expression used is similar to that for the native protein (data not shown). In the closed conformations seen in eIF4A and BstDEAD, Ser67 and Thr50 interact with the residues of the DEAD motif but are positioned too far from the SAT motif. In view of these findings, the differences in side-chain length between Nc-rck/p54 (Asn) and other DEAD-box proteins (Ser or Thr) are believed to have an effect on the characteristic extent of P-loop swiveling.

In previously determined crystal structures, the strictly conserved Lys side-chain of the P-loop is positioned in such a way that it interacts with the first Asp and Glu of the DEAD motif. These two latter residues have been shown to be involved in water-mediated Mg binding and hydrolysis of ATP (Story & Steitz 1992). In Nc-rck/p54, the side-chain of Lys135 is probably involved in such interactions, as it is positioned around 2.8 Å away from the side-chain of Asp235 (Fig. 1C). However, it is positioned too far from the side-chain of Glu236 for interaction, although Glu236 is positioned near Gln131 (3.2 Å) in the P-loop. Moreover, the crystal structures of eIF4A and BstDEAD both have the closed P-loop conformation, demonstrating that similar interactions would stabilize such a conformation of the P-loop. These interactions of Lys135 could thus effectively stabilize the closed conformation of the P-loop as another means of regulating ATP binding and hydrolysis.

The final Asp residue in the DEAD motif does not appear to directly interact with the phosphate of ATP. However, through interactions with the SAT motif, this Asp residue may act as a link between ATP hydrolysis and RNA unwinding. In the Nc-rck/p54, the side-chain of Asp238 is positioned within 2.9 Å and 3.2 Å of the hydroxyl side-chains of Thr268 and Ser266, respectively (Fig. 1D). Furthermore, the side-chain of Asp238 is situated such that it may participate in hydrogen bonding interactions with the main chain of the SAT motif. Mutational data suggest that the specific interactions we observed between the DEAD and SAT motifs may be crucial for coupling the ATPase cycle with RNA duplex unwinding (Pause et al. 1994; Plumpton et al. 1994).

The last glutamine in the Q motif (Glu112 in rck/p54) has a role in recognizing the adenine base of ATP. In the crystal structure of eIF4A having an open P-loop conformation complexed with a nucleotide (Benz et al. 1999), it was noted that the conserved glutamine (Gln48) of the Q motif forms hydrogen bonds with the N6 and N7 of the adenine, while the conserved aromatic group (Phe41) stacks with the base (Fig. 1E). Moreover, Ser45 and Gln48 of the Q motif form interactions with the highly conserved Thr69 and Gly70 residues of the P-loop, respectively. In the structure of Nc-rck/p54, Trp105 (Phe41 in eIF4A) is positioned too far away from the ATP binding pocket (Fig. 1E). Furthermore, Ser109 (Ser45) and Gln112 (Gln48) are also too far from Thr133 (Thr69) and Gly134 (Gly70) to make contact. Instead, Thr133 interacts with Ser136 in motif I to stabilize the greatly swiveled P-loop. This interaction is unique to Nc-rck/p54. Moreover, the highly conserved upstream Phe87 stabilizes the loop of the Q motif by interacting with Pro108 of the Q motif.

Role of the GG motif

In the crystal structures of other DEAD-box proteins (Benz et al. 1999; Johnson & McKay 1999; Story et al. 2001), the region containing the GG motif is too disordered to trace or have relatively high B-factors. Thus, this region is presumed to be very flexible in the molecule. In the B chain of Nc-rck/p54, tartaric ion coordination and residue packing with neighboring molecules stabilized the GG motif (Fig. 1A). The corresponding region in chain A is relatively disordered with average B-factor of 38 Å² as compared to 28 Å² for that of chain B. A tartaric ion interacts with the residues of the GG motif, and with motifs Ia and Ib directly or via bridging water molecules. When we superimposed Nc-rck/p54 on to the counterpart in the crystal structure of HCV NS3 helicase complexed with poly(dU)₈ (Fig. 3A), the possibility that poly(dU)₈ interacts with the GG motif of Nc-rck/p54 was confirmed due to overlapping tartaric ions. The poly(dU)₈ binding site of HCV NS3 helicase and the model of the same region of Nc-rck/p54-bound poly(dU)₈ are shown in Fig. 3B,C. In the HCV NS3 helicase structure, the dU8 phosphate backbone is stabilized by hydrogen bonds with O of Thr269, the main chain NH of Lys272 in motif Ib, and the main chain NH of Gly255 in the GV sequence. The dU7 phosphate forms a hydrogen bond with the Val232 NH and interacts with the Ala233 NH and the Ser231 O via bridging water molecules. On the other hand, in the model for Nc-rck/p54, the corresponding residues in motifs Ia and Ib are situated in positions similar to those in HCV NS3 helicase. The G190 in the GG motif is also close to the dU8 phosphate as in the case of G255 in HCV NS3 helicase. We propose that the GG motif binds to the nucleic acid because of interactions with motifs Ia and Ib, which are involved in nucleic acid binding. These findings suggest that the GG motif of rck/p54 and other DEAD-box proteins directly bind to RNAs.

View larger version (26K): [in this window] [in a new window]   Figure 3  Role of the GG motif in DEAD-box protein. (A) Superimposition of GG motif of Ncr-k/p54 on its counterpart in the crystal structure of HCV NS3 helicase complexed with poly(dU)8. The poly(dU)8 is shown as a stick model, as is the case in (B and C). (B) Interactions between HCV NS3 helicase and poly(dU)8 (PDB code, 1a1v; Kim et al. 1998). Three motifs are shown: motif Ia, purple; motif Ib, pink; and GV motif, corresponding to the GG motif in DEAD-box proteins, blue. (C) RNA binding model through the GG motif. Superimposition of A demonstrated that a tartaric ion, which stabilizes the GG motif in the B chain of Nc-rck/p54, was superimposed on to the dU8 backbone phosphate in poly(dU)8. This model could support hydrogen networks between backbone phosphates and each motif.

DEAD-box proteins including rck/p54 can bind to two substrates, ATP and RNA, at the same time. To examine the radial distribution of rck/p54 after the addition of nucleotides, dynamic light scattering (DLS) studies were carried out. These studies on rck/p54 demonstrated that the addition of nucleotides caused changes in the hydrodynamic radii and the polydispersity of rck/p54, which is standard deviation of the Gaussian model at the monomodal fitting (Table 2). The hydrodynamic radii of DEAD-box protein would be measured too large owing to its sphere, so-called the dumbbell-structure (Caruthers et al. 2000). This is because the hydrodynamic radii from DLS measurement defined by rotating the protein. Consistent to the full-length model of eIF4A (Caruthers et al. 2000; PDB code, 1FUU [PDB] ), whose diameter including both N- and C-terminal domains is approximately 9 nm (Fig. 4), native rck/p54 has wide hydrodynamic radii and polydispersity. Moreover, the linker between two domains of rck/p54 is one residue longer than that of eIF4A. It was observed that the addition of ATP enlarged the hydrodynamic radii and widened the polydispersity. DLS measurement of the mixture with ATP was difficult to perform because ATP induced rck/p54 to aggregate. Subsequent SDS-PAGE analysis and DLS measurement showed that those aggregations were caused by intermolecular interactions among domains in rck/p54. It is believed that the energy from the hydrolysis of ATP is used to separate domains consisting of ATP binding motifs in DEAD-box proteins. On the other hand, addition of either ADP or AMP-PMP, the non-hydrolyzing analog of ATP, decreased the hydrodynamic radii. Interestingly, the addition of AMP-PNP had the greatest reducing effect on polydispersity. Because ADP does not have the -phosphate, it could not complete the interaction between triphosphate and consensus motifs, and thus the addition of ADP could not reduce polydispersity as much as AMP-PNP could. These results are consistent with a previous report (Caruthers et al. 2000), which strongly suggested that some motifs in C-terminal domains, considered to be motifs V or VI, are associated with ATP-binding. Thus, these findings indicate that a proper contact distance between the two domains is essential in successfully holding ATP, and subsequent ATP binding and unwinding of RNA.

View larger version (24K): [in this window] [in a new window]   Figure 4  Comparison of dumbbell structure between DEAD-box proteins. DEAD-box protein binds NTP and/or RNAs between both N- and C-terminal domains. (A) Full-length model of eIF4A (Caruthers et al. 2000; PDB code: 1FUU [PDB] ). This model is composed of 384 amino acids It is suggested that the full-length eIF4A would be a distended molecule in solution because of the 11-residue linker between both domains. (B) Crystal structure of MjDEAD (Story et al. 2001; PDB code: 1HV8 [PDB] ). This form of 363 amino acids is still open form although the state between both domains is closer than A. However, this open form is similar to the closed form because ATP could be put between both domains by the superimposition onto PcrA. Conserved motives which are associated with ATP (Motif Q, I, II, SAT, III, V and VI) are shown in pink.

During IRES-dependent translation, ribosomes are recruited to the IRES, which has a complex structure (Jackson & Kaminski 1995; Hellen & Sarnow 2001). In other words, it is important for this cis-acting element at the 5' UTR to assume unique secondary and tertiary structures in order to perform IRES activity. To confirm if the c-myc IRES RNA was unwound by rck/p54, helicase analysis was carried out using electron microscopy. The micrographs showed that almost all of c-myc IRES RNAs are not unstructured but rather, form several structured segments, which resemble protein domains (Fig. 5). This result is consistent with previous studies on the secondary structure of c-myc IRES (Nanbru et al. 1997; Le Quesne et al. 2001). It was apparent from the electron micrograph that almost all c-myc IRES RNAs were unwound by rck/p54. On the other hand, rsbW RNA (RsbW), which is the substrate that had the weakest effect on ATP hydrolytic activity (Fig. 6), was hardly unwound under the same analytic condition (Fig. 5).

View larger version (207K): [in this window] [in a new window]   Figure 5  Electron micrographs of the association between rck/p54 and c-myc IRES or control RNAs. All reactions for helicase analysis were carried out in 50-µL volume containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM MgCl2, 5 mM 2-mercaptoethanol, 7.5 µg of appropriate RNA, 1 mM ATP and 1.5 µg of rck/p54. Every reaction was incubated for 60 min at 30 °C. The control RNA (No IRES) is Bacillus subtilisrsbW (RsbW) which showed the lowest ATP hydrolysis activity in Figure 3. (No IRES) and (IRES) indicate RNAs reacted in the absence of rck/p54. (No IRES + RCK) and (IRES + RCK) indicate RNAs reacted in the presence of rck/p54.

View larger version (14K): [in this window] [in a new window]   Figure 6  RNA dependent ATPase activity of rck/p54. ATP hydrolysis activity of rck/p54 was evaluated using a direct colorimetric assay (Chan et al. 1986). All reactions of ATP hydrolysis assay were carried out at 37 °C for 90 min in 50-µL volume containing 20 mM Tris-HCl (pH 7.4), 150 mM KCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 2 mM ATP, 3 µg of rck/p54, and 3 µg of various of RNAs: without RNA (No RNA), human c-myc IRES RNA (c-myc IRES), human c-myc mRNA without IRES (c-myc), Bacillus subtilisrsbW mRNA (RsbW) and bovine liver tRNAs (tRNA). The ATPase activity in the reaction without RNA was set at 1. The results are the means of four independent experiments.

View larger version (18K): [in this window] [in a new window]   Figure 7  Cell-free luciferase assay. (A) Plasmid construction for luciferase assay based on the cell free system. The HindIII/BamHI digested DNA fragment of IRES was inserted into Luciferase T7 Control DNA (Promega). (B) The effect of rck/p54 on luciferase translation was examined by the use of IRES-conjunct gene. The luciferase activity was measured as described in Experimental procedures. The results are the means of eight data sets from two independent experiments. A standard deviation calculated from control data sets.

So far, we have tested the rck/p54 expression in various cell lines; however, all the cell lines tested exhibited a good expression of rck/p54. Finally, we could establish stable over-expressants of wild- or mutated-rck/p54 in rck/p54-low expressing HeLa cells at 6 weeks after the transfection. The over-expression of rck/p54 protein and RCK mRNA was confirmed by Western blot and reverse transcription (RT)-PCR analyses, and the level in wild-rck/p54 of HeLa (HeLa/RCK) cells was approximately two-fold greater than that of the HeLa/IRES cells (Fig. 8A,B). The mutated RCK mRNA in HeLa/mRCK cells was detected, but not the protein by Western blot analysis using anti-rck/p54N antibody. The growth curve of HeLa/RCK cells showed an extremely low growth rate compared with that for HeLa/IRES or HeLa/mRCK cells whose growth was slightly inhibited (Fig. 8C). Since the N-terminal core domain and a part of C-terminal domain of rck/p54, which has an RNA binding activity, is produced in HeLa/mRCK cells, a slight growth suppressive effect may be exerted. A morphological study by phase-contrast microscopy revealed the phases that HeLa/RCK cell shaped a large body and had long-branched processes in contrast with HeLa/mRCK or HeLa/IRES cells (Fig. 8D). This finding led us to speculate cell-cycle arrest in HeLa/RCK. To examine how enforced expression of rck/p54 would affect the cell-cycle progression in these cells, we measured their DNA content by fluorescence-activated flow cytometry (FACS) (Fig. 8E). The cell-cycle analysis clearly demonstrated that more HeLa/RCK cells were in a cell-cycle position consistent with the G2/M phase compared with the other transfectants, which may reflect the growth inhibition and morphological changes in the HeLa/RCK cells. In addition, in HeLa/RCK cells, the level of c-myc expression was extremely decreased (Fig. 8A).

View larger version (53K): [in this window] [in a new window]   Figure 8  Enforced over-expression of rck/p54 in HeLa cells. (A) Transfectants characterized by Western blot analysis. The anti-rck/p54M was used for Western blot analysis. (B)RT-PCR of T-RCK. ß-actin was used as an internal control. Lane contents are the same as in (A). (C) Cell growth of the stable transfectants. Viable cell number was counted after inoculating cells into the culture dishes (1 x 105/mL). Data are presented as the mean ± S.D. of three different experiments, each carried out in duplicate. (D) Morphological aspects of the transfectants observed by phase-contrast light microscopy. (a) HeLa cells, (b) HeLa/IRES cells, (c) HeLa/mRCK, (d) HeLa/RCK. (E) Cell-cycle analysis of the transfectants by FACS. The percentage of cells in each phase is indicated. Data representative of those from three independent experiments are shown.

	Discussion

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Rabbit eIF4A has poor substrate specificity and helicase activity in vitro (Rogers et al. 2001) but such activities can be enhanced by the addition of another translation-initiation factor, eIF4B (Ray et al. 1985). Although eIF4A functions within a multiprotein complex involved in translation-initiation (Gingras et al. 1999), rck/p54 is thought to function as a monomer. Members of RCK subfamily have N-terminal extensions that may play an essential role in the enzymatic activity and may not require additional initiation factors. Furthermore, the closed P-loop conformation may play an important role in keeping interactions between the N-terminal extension and the residues within motifs involved in ATP binding. Therefore, the results of mutagenesis on Asn131 are consistent with this speculation.

The DLS study on rck/p54 revealed that ATP binding induced conformational changes (Table 2). Upon the addition of AMP-PNP, the hydrodynamic radii of rck/p54 became smaller and its polydispersity narrower. These strongly suggested that a proper contact distance between both domains is necessary for ATP binding. It is because the distance between conserved residues in both domains should be closer than the opened form of MjDEAD in order to bind ATP successfully (Fig. 4). On the contrary, addition of ATP enlarged its hydrodynamic radii and widened its polydispersity. This indicates that ATP hydrolysis separates the two domains at the ATP binding site. On the other hand, although rck/p54 of 472 amino acids has N- and C-terminal extension in comparison with 384 amino acids of the full-length model of eIF4A (Caruthers et al. 2000; PDB code, 1FUU [PDB] ), the radius of rck/p54 with AMP-PNP was smaller than its model (Table 2). It is suggested that N- and C-terminal extension of rck/p54 would be involved in ATP-induced conformational change. The present study using DSL is the structural demonstration of the dynamics of ATP recognition in DEAD-box proteins, where rck/p54 was shown to utilize ATP for RNA unwinding in a manner consistent with the reported inchworm model (Tanner & Linder 2001).

The results from the ATP hydrolytic assay (Fig. 6) showed that c-myc IRES RNA could easily be bound and unwound by rck/p54 whereas RsbW are not functional. Those results are consistent with results from present helicase assay (Fig. 5). Interestingly, c-myc IRES RNA enhanced ATP hydrolytic activity to a greater extent compared to bovine liver tRNAs, which is well-known for its unique closely packed structure. According to reported molecular models of c-myc IRES RNA (Nanbru et al. 1997; Le Quesne et al. 2001), regions of secondary structure are abundantly included in its models. Thus those results also indicated that rck/p54 could not ubiquitously use all types of structural RNAs for ATP hydrolysis. In addition to the present DLS results, we can conclude that a proper contact distance between the two domains of rck/p54 is necessary to unwind structural RNAs by ATP hydrolysis.

The results from the present ATP hydrolytic assay, the present helicase assay, and in vitro luciferase assay demonstrated that the c-myc IRES RNA was a better substrate for rck/p54 and could be easily unwound by rck/p54 (Figs 5–7). These suggested that the unwinding activity of rck/p54 toward the IRES RNAs would lead to the deterioration of IRES-dependent translation. On the other hand, the effects of enforced over-expression of wild-type rck/p54 on cell growth inhibition and morphological change were rather severe in HeLa cells (Fig. 8). It is certain that excessive rck/p54 causes cancer cells to undergo severe growth suppression. According to the effect of rck/p54 on cell cycle, it was reported that Saccharomyces cerevisisae Dhh1 modulated mRNA metabolism in the recovery from G1/S cell-cycle arrest following DNA damage (Bergkessel & Reese 2004). Since rck/p54-over-expressants failed to express c-myc (Fig. 8), the expression of the proteins under c-myc control could be negatively regulated by rck/p54 (Habel et al. 2005; Ushmorov et al. 2005). These findings altogether suggest that c-myc IRES RNA may lose a functional structure that could recruit the ribosomes to the internal start codon. Thus, it is speculated that the unwinding activity of rck/p54 toward the c-myc IRES RNAs could lead to the deterioration of IRES-dependent translation due to the destruction of its functional structure.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Structure determination and refinement

Expression, purification, crystallization and data collection of Nc-rck/p54 were as previously described (Matsui et al. 2004). Structure was determined by molecular replacement with the CCP4 program MOLREP (Collaborative Computational Project 1994; Vagin & Teplyakov 1998) using the ATPase domain of translation initiation factor 4 A (eIF4A) from Saccharomyces cerevisiae (PDB code: 1QDE [PDB] , Benz et al. 1999) as the search model. The resulting solution had a correlation coefficient of 37.3% and an R factor of 51.8%. The model was improved by simulated annealing followed by energy minimization and B-factor refinement with CNS (Brünger et al. 1998). Xfit/Xtalview (McRee 1999) was used to examine the maps and to build molecular models. The current model consists of amino acids 83-288 from both chains, one Zn²⁺ ion, one tartaric ion and 325 water molecules. Refinement statistics for the current model are summarized in Table 1. Of the non-proline, non-glycine residues, 93.1% lie in the most favored region of the Ramachandran plot with 0% found in the disallowed regions as defined by PROCHECK (Laskowski et al. 1993).

The binding of nucleotides was carried out in 100 µL reaction volumes, containing 2.5 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.5 mM DTT, 0.5% glycerol, 100 mM MgCl₂, 1 mM nucleotide and 185 µM rck/p54 protein. The reactions were incubated on ice for 5 min. The protein-nucleotide solution was then filtered through 0.02 µm Anodisc 13 membrane filters (Whatman) to remove particulates. Dynamic light scattering studies on rck/p54 were carried out using the DynaPro-99 equipment (Protein Solutions Ltd). The number of the acquisition scans was 20 and the acquisition time was 5 s per scan at 20 °C. The data were analyzed using Dynamics v.5.25.44 software.

In vitro transcription c-myc IRES cDNAs, which correspond to the region containing nucleotides 162-562 in the human c-myc (GenBank accession number: V00568; Watt et al. 1983) were synthesized by Takara Bio Inc. The synthesized c-myc IRES is comprised of the region from the P2 promoter to the cytosine nucleotide that exists after the initiator AUG in the c-myc 5' UTR transcript. PCR amplification of c-myc IRES was briefly carried out using the 5' primer containing the T7 polymerase promoter sequence to make the template for in vitro transcription, and then the PCR products were transcribed using the in vitro transcription kit "MEGAscript" (Ambion). These were performed according to manufacturer's protocol.

The plasmid containing c-myc cDNA, which corresponds to the coding region was as previously described (Akao et al. 2003). After linearization with the restriction enzyme, the resulting plasmid was transcribed using the in vitro transcription kit.

The template for firefly luciferase was obtained by PCR from Luciferase T7 control DNA (Promega) using the 5'-containing T7 polymerase promoter primer and 3' primer corresponding to the sequence up to the stop codon. Promega Corporation provided the information on the 3' primer sequence. The 1.7 kb template was also transcribed using the in vitro transcription kit.

The template for Bacillus subtilisrsbW was generated from an EcoRI-digested pSTBlue-1 vector (Novagen), which contains the coding region of rsbW at the blunt cloning site. The rsbW DNA sequence corresponds to the region 55151-55480 in the Bacillus subtilis genome sequence (GenBank accession number: AB001488). This template, whose transcript is 0.4 kb long, was also transcribed using the above-mentioned in vitro transcription kit.

ATP hydrolysis assay

The ATP hydrolytic activity of rck/p54 was evaluated using a direct colorimetric assay by measuring the concentration of the inorganic phosphate (Chan et al. 1986; Huang & Liu 2002). This method utilizes the reaction product of the malachite green-molybdate reagent with the inorganic phosphate, the phosphomolybdate-malachite green complex, which can be quantified spectroscopically. The malachite green-molybdate reagent was made by mixing one volume of 10 mM ammonium molybdate tetrahydrate in 4.5 M HCl and three volumes of 0.0053% (w/v) malachite green and was then incubated for 60 min at room temperature before use.

All reactions of the ATP hydrolytic assay were carried out in 50-µL volumes containing 20 mM Tris-HCl (pH 7.4), 150 mM KCl, 2 mM MgCl₂, 1 mM 2-mercaptoethanol, 3 µg of appropriate RNA, 2 mM ATP and 3 µg of rck/p54. To react completely, every reaction was incubated for 90 min at 37 °C. The bovine liver tRNAs were purchased from Sigma-Aldrich Inc. After incubation, 150 µL of the malachite green-molybdate reagent was added to the reaction, and the mixture was further incubated for exactly 7 min at room temperature. Measurement of the absorption at 630 nm was performed using the UV-1700 spectrophotometer (SHIMADZU) at certain periods of time, and the concentration of the inorganic phosphate from ATP hydrolysis was estimated from the calibration curve that was obtained using potassium dihydrogen phosphate solutions of known concentrations.

Helicase analysis using the electro microgram

All reactions for helicase analysis were carried out in 50-µL volumes containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM MgCl₂, 5 mM 2-mercaptoethanol, 7.5 µg of appropriate RNA, 1 mM ATP and 1.5 µg of rck/p54. Every reaction was incubated for exactly 60 min at 30 °C. After the reaction, up to 50% glycerol was added to the reaction solution. Thirty millilitre of each preparation were sprayed on to a mica surface cleaved freshly by using a painter's airbrush (Olympus Model SP-B, 0.18 mm). Then, the mica was rapidly brought into a freeze-etching device equipped with a large turbo pump (ER 7000, Hitachi), dried for 10 min (room temperature) in a vacuum (1 x 10^-6 Pa), and then cooled to –100 °C. Subsequently, the specimens were rotary shadowed with platinum by an electron gun positioned at an angle of 2.5° to the mica surface, a procedure that was followed by carbon evaporation. Shadowed films were removed from the mica by slowly soaking them in water, and then mounted on copper grids for observation. We found that low-angle rotary shadowing at such a low temperature and strong vacuum enhanced the resolution by reducing the particle size of the evaporated platinum. In the conventional method (Kachi et al. 2000), the temperature is approximately 20 °C and the vacuum was not as strong.

Luciferase assay using cell free system

The synthesis of c-myc IRES cDNA clone, which corresponds with the region 162 to 562 in the human mRNA encoding c-myc oncogene (GenBank accession number: V00568; Watt et al. 1983) was ordered from Takara Bio Inc. The synthesized c-myc IRES is contained in the region from the P2 promoter to a cytosine after the initiator AUG in the c-myc 5' UTR transcript. For in vitro translation, the plasmid including c-myc IRES was constructed as Fig. 7. Briefly, the synthesized c-myc IRES was further amplified with primers with HindIII and BamHI sites, and then inserted to these restriction sites in Luciferase T7 Control DNA (Promega). The firefly luciferase protein translated from this plasmid template has additional four residues, Met-Arg-Ile-Gln, at N-terminus. The luciferase proteins were transcribed and translated from plasmid DNA template using in vitro translation kit "TNT coupled reticulocyte lysate system" (Promega). Based on manufacturer's protocol, the reaction was carried out in 25-µL volume with/without 0.2–2.0 µg of rck/p54. Each reaction was incubated for 30 min at 37 °C. After reaction, reaction mixture was diluted 10 times with the sterile water, and then 20 µL of diluted reaction mixture were added to 100 µL of the luciferase assay regent "Luciferase Assay System" (Promega). Chemiluminescence signal was measured in a luminometer "Turner Designs Luminometer Model TD20/20" (Promega). The results are the means of eight data sets from two independent experiments.

Cell culture, morphological study and cell viability

Human malignant cell line HeLa (uterine cervical cancer) was grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and 2 mM L-glutamine under an atmosphere of 95% air and 5% CO₂ at 37 °C. The morphological study was performed by using phase-contrast microscopy. The number of viable cells was determined by the trypan-blue dye exclusion test.

Plasmid construction and DNA transfection with wild-type or mutant-RCK gene

For establishment of transfectants expressing rck/p54 or its deletion mutant, we used a pIRES1neo eukaryotic expression vector (BD Biosciences Clontech). The pIRES1neo vector, derived from pCIN4, contains the IRES of the encephalomyocarditis virus, and permits the translation of two open reading frames, RCK and neomycin phosphotransferase II cDNA, from one messenger RNA. For construction of the full-length human RCK gene (pIRES-RCK) and its deletion mutant (1–189 amino acids) (pIRES-mRCK), the coding regions were obtained from RCK cDNA clone (Akao et al. 1995) by PCR and then inserted into the BamHI-cleaved pIRES1neo vector. The insert regions of plasmid vectors were confirmed by DNA sequencing. HeLa cells were transfected with pIRESneo, pIRES-RCK or pIRES-mRCK by using liposomes (Lipofect-AMINE) according to the manufacturer's Lipofection protocol (Gibco BRL, Rockville, MD, USA). Briefly, the cells (5 x 10⁵ cells/60-mm dish) were cultured for 24 h and then incubated with liposome-entrapped pIRES1neo, pIRES-RCK, or pIRES-mRCK (1 µg DNA/100 nmol of lipids in 1 mL of the medium). After incubation for 16 h, the cells were cultured in fresh medium for 1 day and then selected with geneticin (G418; Nakarai, Kyoto, Japan) at the concentration of 750 µg/mL in the medium, which was exchanged for fresh G418-containing medium every 3 days. The living cells were then segregated by limiting dilution, and individual clones were isolated. The stable clones over-expressing rck/p54 (HeLa/RCK) or mutant rck/p54 protein (HeLa/mRCK) were confirmed by RT-PCR and Western blot analyses. The control clone, which contained the pIRESIneo vector alone, was also isolated and named HeLa/IRES.

Semi-quantitative RT-PCR

Total RNA was isolated by using an RNAqueous-4PCR kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. RNA samples were reverse-transcribed by using Super Script II RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo(dT) primer (Invitrogen). Prepared cDNA samples were purified by use of a PCR Purification kit (Qiagen, Hilden, Germany) and used for PCR. The PCR was performed according to the manufacturer's instruction. ß-actin was used as an internal control. Primers for RCK and c-myc were as follows: T-RCK forward, 5'-GGCTGGGAAAAGCCATCT-3'; T-RCK reverse, 5'-ACCTGATCTTCCAATACG-3'; c-myc forward, 5'-ACATCATCATCCAGGACTG-3'; c-myc reverse, 5'-TTTAGCTCGTTCCTCCTCTG-3'. The PCR products were evaluated by agarose electrophoresis.

Western blotting

The cells were homogenized in chilled lysis buffer comprising 10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 1% Protease Inhibitor Cocktail (Sigma) and stood for 30 min on ice. After centrifugation at 14 000 r.p.m. for 20 min at 4 °C, the supernatants were collected as protein samples. Protein contents were measured with a DC Protein assay kit (Biorad, Hercules, CA, USA). One microgram of lysate protein for Western blot of rck/p54 was separated by SDS-PAGE using a 12% polyacrylamide gel and electroblotted on to a polyvinylidene fluoride membrane (PVDF) (Du Pont, Boston, MA, USA). After blockage of nonspecific binding sites for 1 h with 5% non-fat milk in PBS containing 0.1% Tween 20, the membrane was incubated overnight at 4 °C with anti-human rck/p54N antibody (Matsumoto et al. 2005). For Western blotting of c-myc, 10 g of lysate protein was used. The membranes were then washed 3 times with PBS containing 0.1% Tween 20, incubated further with horseradish peroxidase (HRP)-conjugated sheep anti-mouse or donkey anti-rabbit Ig antibody (Amersham Biosciences, Piscataway, NJ, USA) at room temperature, and then washed 3 times with PBS containing 0.1% Tween 20. The immunoblots were visualized by use of an enhanced chemiluminescence detection kit (New England Biolabs, Beverly, MA, USA).

Cell-cycle analysis

After culture for 36–48 h, cells were trypsinized and washed twice in ice-cold PBS. The cell pellets were resuspended in 300 µL of 1% FBS/PBS and fixed for 15 min at 4 °C by the addition of 700 µL of 100% ethanol. The fixed cells were washed and resuspended in 500 µL of PBS containing 2.5 µL of RNase. The cells were incubated at 37 °C for 15 min, and DNA content was determined by the addition of 50 µL propiodium iodide (Sigma). Cell-cycle analysis was performed FACS using a FACScaliber and CellQuest software (Becton Dickson, San Jose, CA, USA).

	Acknowledgements

 
We thank Mr Wataru Adachi and Professor Akio Takénaka (Tokyo Institute of Technology) for their help with the dynamic light scattering measurements. This study was supported in part both by a grant from the National Project on Protein Structural and Functional Analyzes and by a Grant-in-Aid for Scientific Research (to Y.A., no. 14657061) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This study was also supported by a grant from SPring-8.

	Footnotes

 
Communicated by: Kozo Kaibuchi

Present address: ^aDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB-31, La Jolla, CA 92037, USA

^* Correspondence: E-mail: yakao{at}mail.giib.or.jp, tkumasak{at}bio.titech.ac.jp

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Received: 24 October 2005
Accepted: 17 January 2006

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