The Isolation and Characterization of an RNA Helicase from Nuclear Extracts of HeLa Cells*

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electropho- resis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nu- cleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/ DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3‘ to 5’ and the 5’ to 3’ directions. The of the RNA helicase here unwinding activity and then dried, and the regions corresponding to the mono-, di-, and triphosphate derivates were located by UV illumination, excised, and quantitated by scintillation counting. One unit of NTPase catalyzed the RNA-dependent hydrolysis of 1 pmol of NTP under the conditions de- scribed above. Protein Assay-Protein concentration was determined using the Bio-Rad protein assay reagent with BSA as the standard. Protein analysis by SDS-polyacrylamide gel electrophoresis (4% acrylamide stacking gel, 10% acrylamide resolving gel) was carried out as described by Laemmli (25). Protein bands were visualized using the ICN rapid silver stain kit.

their translation efficiency. Other well-characterized RNA helicases include the human p68 protein ( 5 ) and the SV40 Tantigen (6). Recently, the cylindrical inclusion (CI) protein encoded by the plum pox virus (PPV), which is a positive ssRNA virus, was shown to contain RNA helicase activity (7-9). It has been suggested that this protein is involved in the RNA replication reaction. All of these enzymes, in addition to displacing dsRNA also contain RNA-dependent nucleoside triphosphatase activity. In addition to eIF-4A, p68, and the CI protein of PPV, a large group of proteins possess strong sequence homology to proteins containing RNA helicase activity. However, it is not known whether they catalyze the displacement of duplex RNA. These proteins include the yeast genes translation initiation factors 1 and 2 and MSS116 (10, l l ) , t h e Escherichia coli SrmB protein (which contains RNAdependent ATPase activity) (12), the Drosophila uma gene (13), the mouse PLlO gene (14), and some of the newly discovered yeast prp genes (15,16), which play important roles in pre-mRNA splicing. Whereas the precise role of a number of these proteins is unknown, their action must be related to RNA metabolism. There is genetic evidence that the yeast mutant MSS116 affects mitochondrial RNA splicing (10).
ATP is required at multiple stages in the assembly of spliceosomes and the splicing of pre-mRNA in vitro in both yeast and mammalian systems (17)(18)(19)(20). No other nucleoside triphosphate can replace this requirement (21). The specific reactions that require ATP in the assembly of spliceosomes are presently unknown. However, the role of RNA helicases, which are capable of altering the interactions between dsRNA both intramolecularly and intermolecularly, would be well suited for the sequential steps known to involve interactions between RNAs. An excellent example for an important role of an RNA helicase is in the displacement of the U4/U6 RNAs. There is strong evidence that the U4/U6 small nuclear ribonucleoprotein is a single nucleoprotein particle in which U4 and U6 RNAs are hydrogen-bonded together (22). During the formation of the spliceosome, these RNAs are displaced, and U4 RNA is released from the complex while U6 RNA is retained (18,23).
Prompted by these considerations, we have initiated the purification and characterization of RNA helicases present in nuclear extracts of HeLa cells. We have detected a variety of different RNA helicases; and in this paper, we describe the purification and the properties of one of these enzymes. This enzyme, in the presence of nucleoside triphosphates, displaced a variety of partial dsRNAs, including the U4/U6 RNAs, and is specific in its requirement for partial dsRNA. RNA/DNA hybrids as well as duplex DNA structures were not displaced.  The mixtures were incubated a t 40 "C for 2 h, and the RNA products were extracted twice with phenol and once with chloroform/isoamyl alcohol (24:1), treated with ethanol (2.5 volumes), and then centrifuged for 30 min in an Eppendorf microcentrifuge. The pellets were dried under vacuum for 20 min. The transcripts were resuspended in a solution containing 90% formamide, TBE buffer (Tris, 0.089 M, borate, 0.089 M, EDTA, 0.002 M) 0.02% bromphenol blue, and 0.02% xylene cyanol and loaded onto a 10% polyacrylamide, 8 M urea denaturing gel. Electrophoresis was carried out at 20 mA for 45 min, and the RNA products were located by placing the gel on top of a TLC cellulose plate illuminated with UV light. The regions of the gel containing the RNA transcripts were excised and ground with a glass rod in a 1.5-pl Eppendorf tube with 0.4 ml of a solution containing 0.5 M ammonium acetate (pH 7.0), 0.1% SDS, and 10 mM EDTA. The mixture was incubated for 2 h a t 4 "C with gentle agitation and centrifuged for 2 min, and the supernatant was then precipitated with 2.5 volumes of ethanol. After 1 h at -80 "C, the suspension was centrifuged for 30 min, the alcohol was removed, and the pellet was dried under vacuum for 20 min. The RNAs were dissolved in 0.1 ml of buffer T (40 mM Hepes (pH 7.0), 0.1 M NaQO.5 mM EDTA, 0.002% Nonidet P-40). For the preparation of partial dsRNAs, equimolar amounts (50-150 pmol) of each transcript were mixed together and diluted to 0.2 ml with distilled water (if necessary), and 50 p1 of 5 X hybridization buffer (2.5 M NaC1, 0.2 M Hepes (pH 7.0), 5 mM EDTA) was added. The hybridization mixture was incubated at 95 "C for 5 min and immediately switched to a 55 "C water bath and incubated overnight at this temperature. The annealed RNAs were precipitated with ethanol as described before, resuspended in 20 pl of loading buffer (0.025% bromphenol blue, 0.25% xylene cyanol, 0.05% SDS, 1 mM EDTA, and 15% glycerol), and loaded onto an 8% polyacrylamide nondenaturing gel. The RNAs were electrophoresed at 6 mA for 2-3 h, and the duplex RNA band was located by autoradiography. In all experiments, control lanes with nonhybridized transcript were carried out for comparison. The gel slice containing the duplex RNA was eluted as described above. The partial dsRNA was precipitated with ethanol and resuspended in buffer T a t a concentration of 50 fmol/pl. Such RNA substrates had a specific activity of 1500-2000 cpm/fmol and could be used for as long as 1 month.

Preparation of R N A Helicase
To test the directionality of the RNA helicase, 3'-and 5"tailed substrates were prepared (Fig. 1B). The 5"tailed substrate was prepared by transcription of the pSP65 plasmid (cut with XbaI) in the presence of rNTPs with [a-:"P]GTP and the pGEM3 plasmid (cut with KpnI) in the presence of rNTPs with ["HIGTP. The transcription reactions, purification, and annealing of the transcripts were as described above. The 3"tailed substrate was prepared by transcription of the pGEM3-CS-plasmid as described by Scheffner et al. (6). The short strand was prepared by T 7 RNA polymerase transcription of the pGEM3-CS-plasmid (cut with RsaI) in the presence of rNTPs with [a-:"P]GTP. The complementary strand was prepared by transcription with SP6 RNA polymerase of the pGEM3-CS-plasmid (cut with HaeII) in the presence of rNTPs with ["HIGTP. The transcription reaction, purification, and annealing of the transcripts were as described above. The specific activity of each substrate was 1000 cpm/fmol. double-stranded regions and the calculated T , values are indicated.C, U4/U6 partial dsRNA substrate. The substrates were prepared as described under "Materials and Methods." The sequences of the yeast U4 and U6 RNAs, the calculated T,, and the proposed structure of the dsRNA substrate are indicated. The yeast U4/U6 RNAs are hydrogen-bonded together by 21 intermolecular base pairs. D, DNA/ DNA and RNA/DNA substrates. The substrates were prepared as described under "Materials and Methods." The 30-nucleotide synthetic oligonucleotides (complementary to nucleotides 5127-5156 of 4x174 circular ssDNA) were annealed to 6x174 DNA as described under "Materials and Methods." The complementary sequence, the calculated T,, and the structure of the substrates are shown.
The yeast clones of U4 and U6 small nuclear RNAs were a generous gift of Drs. P. Fabrizio and J. Abelson (California Institute of Technology) and were originally described by Siliciano et al. (24). The U4/ U6 partial dsRNA substrate was prepared in a similar way as described above, except that both the plasmid containing the yeast U4 RNA sequence (cut with StyI) and the plasmid containing the yeast U6 RNA sequence (cut with RsaI) were transcribed with T7 RNA polymerase in the presence of rNTPs with [w~'P]GTP as described above, except for the hybridization, which was carried out at 40 "C. Purification of the RNA transcripts and the duplex RNA was as described above. The specific activity of the U4/U6 partial dsRNA substrate was 1000 cpm/fmol. The DroDosed structure of the U4/U6 dsRNA (22) is depicted 'in Fig. 1C.
" DNA/DNA and RNA/DNA substrates (Fig. 1D) were DreDared bv labeling the 5'-end of a synthetic 30-nucleot2e deoxyoliionhcleotide or oligoribonucleotide of the same sequence with polynucleotide kinase and [y3'P]ATP. The labeled oligonucleotides were purified by gel electrophoresis and annealed to 4x174 circular ssDNA as described above, except that the hybridization was carried out at 45 "C in a volume of 50 pl (overlaid with light mineral oil). The duplex structures were loaded onto 0.5-ml Sepharose 4B columns and eluted with buffer T. The early eluting fractions, containing duplex structures separated from the free oligonucleotide, were pooled and directly used for helicase assays. The specific activity of each substrate was 800 cpm/fmol.
Helicuse Assay-Unless otherwise indicated, the assay for RNA helicase was carried out in reaction mixtures (20 pl) containing 40 mM Tris-HC1 (pH 7.5), 1 mM MgC12, 3 mM ATP, 2 mM DTT, 0.15 M NaC1,50 pg/ml BSA, 2-5 units of RNasin, 50 fmol of the 3ZP-labeled RNA substrate, and the helicase fraction. All reactions were assembled on ice and then incubated at 30 "C for 20 min. Reactions were stopped by adding 4 pl of a loading solution (25% (v/v) glycerol, 0.5% SDS, 50 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol, and 0.2 mg/ml proteinase K), followed by a 15-min incubation at room temperature. Half of each sample (12 pl) was loaded onto a 17% polyacrylamide gel and electrophoresed at 10 mA for 2-3 h. The gels were dried and autoradiographed, and the labeled bands were excised and quantitated by scintillation counting. One unit of RNA helicase catalyzed the displacement of 1 pmol of RNA under the above conditions. Background values (ssRNA) represented 4 % of the input dsRNA. This value has not been subtracted from the results presented.
dTTPuse Assay-The measurement of dTTP cleavage was carried out in reaction mixtures (20 pI) containing 40 mM Tris-HC1 (pH 7.5), 1 mM MgC12, 2 mM DTT, 1 mM poly(A) (as nucleotides), 10 p M [3H] dTTP (400-500 cpm/pmol), and the enzyme fraction. To determine ATPase or GTPase activity, [3H]dTTP was replaced by [3H]ATP or ["HIGTP, and the assay conditions were identical. Mixtures were incubated at 30 "C for 60 min, after which time 2 pl of each reaction was spotted onto a polyethyleneimine-cellulose plate containing 0.02 pmol each of the nucleoside mono-, di-, and triphosphates of the labeled nucleotide species as markers. The plates were developed with a solution containing 1 M HCOOH and 0.5 M LiCl and then dried, and the regions corresponding to the mono-, di-, and triphosphate derivates were located by UV illumination, excised, and quantitated by scintillation counting. One unit of NTPase catalyzed the RNAdependent hydrolysis of 1 pmol of NTP under the conditions described above.
Protein Assay-Protein concentration was determined using the Bio-Rad protein assay reagent with BSA as the standard. Protein analysis by SDS-polyacrylamide gel electrophoresis (4% acrylamide stacking gel, 10% acrylamide resolving gel) was carried out as described by Laemmli (25). Protein bands were visualized using the ICN rapid silver stain kit.

Preparation of HeLa RNA Helicase
Ammonium Sulfate Fractionation-Dignam extract was prepared from HeLa cells as previously described (26). The nuclear extract (350 ml, 6 mg/ml protein) obtained from 4.5 X 10" cells was brought to 60% ammonium sulfate saturation by adding solid ammonium sulfate (39 g/100 ml) with continuous stirring at 4 "C for 30 min, and the mixture was then centrifuged at 10,000 rpm for 15 min. The supernatant was removed, and the pellet was washed with a solution containing 60% ammonium sulfate in buffer A (20 mM Tris-HC1 (pH 7.5), 0.5 mM EDTA, 0.01% Nonidet P-40, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol) plus 0.1 M KC1. After centrifugation, the pellet was suspended in a solution (40 ml) containing 40% ammonium sulfate in buffer A plus 0.1 M KCl. After centrifugation, the pellet was reextracted with the same buffer. The supernatants were pooled and adjusted with solid ammonium sulfate to 60% saturation. After centrifugation, the pellet was saved as the ammonium sulfate 6040 fraction. The pellet obtained after the extraction with 40% ammonium sulfate was extracted with a solution (40 ml) containing 30% ammonium sulfate in buffer A plus 0.1 M KC1 and then centrifuged; the pellet was re-extracted with the same buffer and again centrifuged. The supernatants were pooled and precipitated by addition of solid ammonium sulfate to 40% saturation. After centrifugation, the pellet was saved as the ammonium sulfate 40:30 fraction. The pellet obtained after the extraction with 30% ammonium sulfate was suspended in a solution (40 ml) of 20% ammonium sulfate in buffer A plus 0.1 M KC1 and then centrifuged. The pellet obtained was re-extracted with the same buffer. The supernatants obtained after washing with 20% ammonium sulfate were pooled and precipitated by addition of solid ammonium sulfate to 30% saturation. After centrifugation, the pellet was saved as the ammonium sulfate 30:20 fraction. The final remaining pellet was the ammonium sulfate 20:O fraction.
All ammonium sulfate pellets were dissolved in the minimum possible volume of buffer A plus 0.1 M KC1 (-20 ml), dialyzed against three changes of buffer A plus 0.1 M NaCl (1 liter each) for a total of 6 h at 4 "C, and then assayed for RNA helicase activity. The ammonium sulfate 3020 fraction contained the bulk of the enzyme activity and was used for subsequent steps. ssDNA-Cellulose Chromatography-The ammonium sulfate 30:20 fraction (20 ml) was passed through a ssDNA-cellulose column (21 X 1.7 cm', 35 ml, 5-6 mg of DNA/g of resin) previously equilibrated with buffer A plus 0.1 M NaC1. After washing with 50 ml of the same buffer, the column was successively step-eluted with 50 ml each of buffer A plus 0.25, 0.5, and 1.0 M NaCl. Fractions (5 ml) were collected and assayed for both RNA helicase and dTTPase activities. Although the 0.1, 0.25, and 0.5 M salt steps contained RNA helicase with comparable amounts of RNA-dependent dTTPase activity, the material eluted with 0.5 M NaCl contained RNA helicase with the highest specific activity (Fig.  2). This fraction was used for further purification.
CM-Sepharose Chromatography-The 0.5 M NaCl DNAcellulose fraction (42 ml) was dialyzed against three changes (500 ml each) of buffer C (20 mM MES (pH 6.0), 0.5 mM EDTA, 0.01% Nonidet P-40, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol) plus 0.1 M NaC1. Buffer changes were carried out after dialysis for 90 min at 4 "C. The dialyzed fraction was loaded onto a CM-Sepharose column (15 X 1.7 cm', 25 ml) previously equilibrated with buffer C plus 0.1 M NaC1. The column was washed with 1 volume of buffer C plus 0.1 M NaCl and then eluted with 160 ml of a linear gradient of buffer C from 0.1 (80 ml) to 1.0 (80 ml) M NaCl, and 3-ml fractions were collected. Helicase activity was recovered in the material that eluted between 0.3 and 0.4 M NaCl. There was poor correspondence between RNA-dependent dTTPase and helicase activities, and multiple peaks of dTTPase and RNA helicase were detected. Fractions 37-41 were pooled and used for the next step.

FIG. 2.
Elution of RNA helicase and RNA-dependent dTTPase activities from ssDNA-cellulose. The protein concentration and the helicase and RNA-dependent dTTPase activities of the fractions eluted at different salt concentrations are shown. This chromatographic procedure as well as the assays used were as described in the text. cutoff) and concentrated by overlaying solid polyethylene glycol 20,000 to 2.5 ml. The concentrated fraction was dialyzed against two changes (500 ml each) of buffer D (20 mM Tris-HC1 (pH 7.5), 0.5 mM EDTA, 0.01% Nonidet P-40, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride and 20% (v/v) glycerol) plus 1.0 M NaCl for 2 h at 4 "C. This material was then layered onto a 30-ml 0-15% (w/v) sucrose gradient in buffer D plus 1.0 M NaC1. A second identical gradient was loaded with protein molecular size markers. Both gradients were centrifuged at 26,000 rpm in a Sorvall AH641 rotor for 75 h at 4 "C. The tubes were punctured, and the fractions (1 ml) collected from the bottom were assayed for RNA helicase and RNA-dependent dTTPase activities. The leading edges of the peak of dTTPase and RNA helicase activities were the same, but the trailing edges differed significantly. The fractions containing RNA helicase were pooled as SG1 (fractions 20-22), concentrated with solid polyethylene glycol, dialyzed as described above, and then subjected to a second sucrose gradient centrifugation step as described above. After the second sucrose gradient procedure, there was a closer correspondence between the RNA helicase and RNA-dependent dTTPase activities (Fig. 3). Fractions 19-21 were pooled and dialyzed against two changes of 500 ml of buffer D plus 0.2 M NaCl. Unless specified, this fraction (SG2) was used for all of the experiments described here.

Comments on Purification Procedure and Properties of Helicase Activity
The purification procedure summarized in Table I resulted in a 300-fold enrichment of the RNA helicase activity with a yield of 1%. The relatively low yield reflected the presence of multiple RNA helicase activities in fractions that were not included in the various steps developed. We have found that the multiple peaks of RNA helicase and RNA-dependent dTTPase activities (Fig. 2) represent other distinct RNA helicases, some of which have been characterized, whereas others remain to be further studied (data not shown).
The RNA helicase and RNA-dependent dTTPase activities cosedimented in the second sucrose gradient step (Fig. 3). Gel filtration chromatography and sucrose gradient centrifugation, both in the presence of l M N$1, indicated that the helicase has a Stokes radius of 44 A and a sedimentation coefficient of -5.4 S.' Assuming a partial specific volume of 0.725 cm3/g, a native molecular mass of 100 kDa and a frictional ratio of 1.43 can be calculated (data not shown). The various pools containing RNA helicase activity were subjected to SDS-PAGE analysis (Fig. 4, upper), as were the individual fractions collected from the second sucrose gradient (Fig. 4, lower). As shown in Fig. 3, RNA helicase activity was detected in the second sucrose gradient between fractions 18 and 23, with the peak of activity in fractions 20 and 21. The only protein bands detected that co-migrated with the helicase activity were bands of 80 and 55 kDa, which were also present in the first sucrose gradient fraction (Fig. 4, upper). Whether both protein bands together represent the RNA helicase activity is presently unknown.
The final preparation of RNA helicase (SG2 fraction) was stable for at least 6 months. Preparations stored as small aliquots at -80 "C could be freeze-thawed at least four times with no loss of activity.
' This study was carried out in buffers containing Nonidet P-40.
The influence of this nonionic detergent on the Stokes radius is unknown since we do not know whether the helicase binds Nonidet P-40.

Requirements for RNA Helicase Activity
The displacement of RNA from the partial dsRNA was completely dependent on ATP and Mg2+ (Table 11). The RNA helicase activity was heat-labile, sensitive to N-ethylmaleimide treatment, inactivated by proteinase K digestion, and unaffected by micrococcal nuclease treatment. These results indicated that the RNA helicase activity was a protein containing essential SH groups, and RNA (or DNA) did not play

TABLE I1
Effect of various agents on RNA helicase activity RNA helicase assays were carried out as described in the text with omissions and additions as indicated. Experiments 1-3 were separate experiments in which each reaction contained 20 ng of the SG2 fraction. All reactions were incubated a t 30 "C for 20 min, and aliquots were loaded onto a 17% polyacrylamide gel. After autoradiography, the amount of unwound product was quantitated. a role in the catalytic activity of the helicase. The displacement reaction was slightly stimulated by the addition of ssDNA-binding proteins (SSB) isolated from HeLa cells and from E. coli (Table 11).
The RNA helicase activity was supported by all eight common nucleoside triphosphates (Fig. 5 ) . ATP and GTP were the most efficient, whereas dTTP and dCTP were the least effective, and the ATP analogs ATP-yS and AMP-PNP did not support dsRNA unwinding. Nucleoside triphosphate concentrations >1 mM were found to be saturating (data not shown). With the purified RNA helicase fraction, the apparent K, for ATP was -100 PM, whereas the K, for Mg' was 50 PM. The helicase activity was stimulated %fold by NaCl (Table 11), and maximal activity was observed at salt concentrations between 75 and 250 mM NaCl. In the presence of 0.3 M NaCI, the reaction was reduced 70%. The optimal pH was broad, ranging between 6.5 and 9.2, with 50% activity remaining at pH 6.0 and 10.0 (data not shown).

RNA Displaced
In the presence of low levels of RNA helicase (5 ng, equivalent to 50 fmol, assuming 100% purity and that all molecules were active), the rate of displacement was linear for -20 min, after which time the rate decreased. After 60-80 min of incubation, 8 fmol of RNA was displaced (Fig. 7, upper).
Addition of more RNA substrate (another 50 fmol) a t 40 min did not significantly alter the rate of RNA displacement. Addition of more helicase (5 ng) after 40 min of incubation resulted in another burst of activity, indicating that the helicase was either inactivated or sequestered during the reaction.
The addition of the E. coli SSB or the multisubunit HSSB had no effect on the RNA displacement reaction described in Fig. 7 (upper).
In the presence of limiting amounts of RNA substrate (5 fmol), 50 ng (500 fmol) of RNA helicase quantitatively unwound the partial dsRNA (Fig. 7, lower). After 15 min of incubation, the addition of more RNA helicase had no further effect, in keeping with the quantitative displacement of the dsRNA. In contrast, the addition of 10 fmol of RNA substrate at 15 min resulted in a rapid burst of helicase activity, which plateaued after quantitative displacement of the duplex RNA. In these reactions, the yield of s R N A was 70% of the expected value based on the input "P-labeled RNA substrate. The reasons for this discrepancy are not clear.

Displacement Polarity of RNA Helicase
The RNA helicase efficiently displaced both 3'-and 5'tailed RNA substrates that lacked any fork structure (Fig. 8). This suggested that the helicase displaced partial dsRNA in both the 5' to 3' and the 3' to 5' directions. The 3'-and 5'tailed substrates were displaced more efficiently than the s20 E were carried out as described in the text in the presence of 5 ng of the RNA helicase SG2 fraction and 50 fmol of the standard RNA substrate. All reactions were carried out in triplicate; to one set, at the time indicated (40 min), more RNA helicase was added. The second set received more RNA substrate (50 fmol) at that time, and the third set received no further supplementation. The reactions were further incubated as indicated. Lower, reaction mixtures contained 5 fmol of RNA substrate and 50 ng of helicase. After 15 min of incubation, one set received an additional 50 ng of helicase, another set was supplemented with 10 fmol of substrate, and the third set received no further addition. Reactions were further incubated as indicated, and the amount of ssRNA formed was determined by gel electrophoresis. standard RNA substrate, probably due to the smaller size (5'tailed substrate) and the lower G + C content (3"tailed substrate) of their dsRNA regions (see Fig. 1). The SV40 Tantigen displaced only the 3'-tailed RNA substrate and not the 5'-tailed dsRNA in the presence of UTP (data not shown).
Scheffner et al. (6) have previously shown that the SV40 Tantigen displaced RNA only in the 3' to 5' direction, which is the same polarity observed with DNA.

Activity of RNA Helicase with Other Duplex Structures
The purified RNA helicase activity specifically displaced dsRNA under the conditions used (Fig. 9). Using the standard partial dsRNA substrate, helicase fractions displaced -35% of the substrate in the presence of ATP (Fig. 9, upper). 4x174 circular ssDNA, hybridized to either a 30-nucleotide complementary oligodeoxyribonucleotide (Fig. 9, center) or the identical 30-nucleotide oligoribonucleotide, did not support helicase activity (Fig. 9, lower). The SV40 T-antigen, in the presence of ATP, displaced both the RNA and the DNA hybridized to the 4x174 circular ssDNA (Fig. 9, center and  lower). Also shown in Fig. 9 (upper), the SV40 T-antigen did not displace the dsRNA in the presence of ATP, but did so in the presence of UTP, in accord with the results of Scheffner et al. (6).
In the generation of the spliceosome complex, it has been reported that the U4/U6 small nuclear ribonucleoprotein coupled to the 55 S complex undergoes a displacement reaction such that only U6 RNA remains associated with the spliceosome (18, 23). To check whether the RNA helicase described here could carry out this displacement reaction, we synthesized the yeast U4 and U6 RNAs both labeled with [a-'"PIGTP from plasmid templates provided by Dr. J. Abelson. The proposed structure of the RNA duplex is described in Fig. 1C. When this RNA, which contains two distinct partial duplex regions, was incubated with the RNA helicase, the RNAs were efficiently separated in the presence of a number of different NTPs added (Fig. 10). Similar results were obtained with the SV40 T-antigen in the presence of UTP (data not shown). in the text with 20 ng of the SG2 fraction and 50 fmol of the duplex substrate, and reaction mixtures were incubated at 30 "C for 20 min. Reactions (20 pl) with the SV40 T-antigen (SV40 T ag) containing 40 mM creatine phosphate (di-Tris salt, pH 7.6), 7 mM MgClZ, 2 mM DTT, 3 mM NTP, and 0.8 pg of the SV40 T-antigen were incubated a t 37 "C for 30 min. Control reactions (-ENZ, no enzyme; A', substrate boiled for 10 min) for each substrate are also indicated. Upper, the substrate used was the standard partial dsRNA; center, the substrate used was the 30-nucleotide oligodeoxynucleotide hybridized to $X174 DNA; lower, the 30-nucleotide oligoribonucleotide was hybridized to @X174 DNA. The enzyme and the nucleoside triphosphate used in each assay are indicated above each lane. An aliquot of each reaction was electrophoresed on a 17% acrylamide gel. After autoradiography of the dried gels, the regions corresponding to the duplex and unwound substrates were excised and quantitated. The amount of s R N A formed is indicated below each lane.

RNA Helicase Has No Deaminase Activity
The displacement of dsRNA due to the deamination of adenine residues has been reported by Bass and Weintraub (27). The generation of inosines in the RNA resulted in the decreased stability of the duplex regions. We have shown that the RNA helicase described here does not alter the A residues in the RNA substrates as well as in the displaced strands. For this purpose, the standard duplex RNA was prepared with [a-'lP]ATP in place of [a-"PIGTP. We found that the unwinding of the AMP-labeled RNA substrate was dependent on the presence of ATP and Mg2+ (in contrast to the Xenopus oocyte displacement system). The unwound RNA and the partial dsRNA were digested with P1 nuclease, and the resulting Reactions were incubated a t 30 "C for 20 min, and aliquots were electrophoresed on a 14% acrylamide gel. After autoradiography of the dried gel, the regions corresponding to the ds-and ssRNAs were excised and quantitated. The amount of ssRNA unwound is indicated below each lane. In these reactions, both U4 and U6 RNAs were labeled as described under "Materials and Methods."

TABLE 111 Effect of different polynucleotides on dTTPase activity
Assays were carried out as described in the text with the polynucleotides in the amounts indicated. Each reaction contained 10 ng of the SG2 fraction and was incubated a t 30 "C for 60 min. Aliquots were loaded onto polyethyleneimine-cellulose plates; and after chromatography, the amount of dTDP formed was quantitated. Churucterizution of $sRNA-dependent Nucleoside Triphosphatase Activity As shown in Fig. 3, both RNA helicase activity and the poly(A)-dependent hydrolysis of dTTP cosedimented in sucrose gradients. Other experiments, such as the rate of heat inactivation at 50 "Cy insensitivity to micrococcal nuclease, and N-ethylmaleimide sensitivity, also supported the conclusion that these two activities were catalyzed by the same protein (data not shown). In the absence of ssRNA, little or no hydrolysis of dTTP (and other NTPs) was detected. In general, ssRNAs supported the hydrolysis of NTPs, whereas dsRNAs were marginally active. A variety of other polynucleotides were examined, as shown in Table 111. The ssRNAs used (poly(A), poly(U)) were active effectors, whereas poly(&) and other ss-or dsDNAs were virtually inactive.
The ssRNA-dependent dTTPase activity was chosen as an assay to follow the RNA helicase, even though dTTP was the least efficient nucleotide that supported the RNA helicase activity. This was done to avoid interference with other more prevalent common NTPase-contaminating activities (ATPase, GTPase) during the purification procedure. However, the level of RNA-dependent dTTPase present in the SG2 fraction was somewhat higher than the triphosphatase activity observed with ATP or GTP (Table IV). The displacement of dsRNA, however, was much lower with dTTP than with ATP or GTP (Fig. 5). This suggests that the requirements for binding RNA and for both binding and breakage of hydrogen bonds differ.

DISCUSSION
The results presented here describe the isolation of an enzyme activity that unwinds partial dsRNA in the presence of Mg2C and a nucleoside triphosphate. This RNA helicase activity has been purified at least 300-fold from HeLa nuclear extracts, which may be an underestimation due to the presence of multiple RNA helicase activities in crude fractions. During the purification of this activity, at least six other peaks of RNA helicase activity were detected that differed in their chromatographic properties. Some of these were evident in Fig. 2. We have further characterized only two of these and found that they have distinct properties from the RNA helicase activity described here (data not shown). We have named the activity in the SG2 fraction RNA helicase I, and we will refer to it as such below.
Preparations of RNA helicase I contain two major polypeptides of 80 and 55 kDa that cosedimented with helicase activity in sucrose gradients carried out in the presence of high salt (1 M NaC1). Furthermore, these two polypeptides also coeluted with RNA helicase I activity when subjected to gel filtration in the presence of 1 M NaCl (data not shown). Whereas these observations suggest a strong interaction between these two polypeptides, we do not know whether both polypeptides are essential for RNA helicase I activity.
RNA helicase I specifically displaced dsRNA and not double-stranded RNA/DNA or DNA/DNA substrates. RNA helicase I is capable of unwinding substrates with either singlestranded 3'-or 5'-regions, suggesting that the enzyme can move bidirectionally on RNA. There are a number of possible explanations for helicase's bidirectionality. These include the following: (i) Bidirectionality is an intrinsic property of RNA helicase I. (ii) The direction of unwinding is modulated by different forms of the same enzyme. (iii) The possibility exists that more than one RNA helicase is present in the SG2 fraction. (iv) The enzyme can displace fully dsRNA structures and does not require a single-stranded tail for translocation. If explanation iv were the case, it would be difficult to determine the direction of translocation. However, RNA helicase I did not displace dsRNA without single-stranded tails (data not shown); and for this reason, we believe that the helicase does require a ssRNA tail for its activity. Bidirectional unwinding has previously been reported only for eIF-4A, which requires eIF-4B for helicase activity. RNA helicase I differs from eIF-4A plus eIF-4B in that it utilizes a variety of nucle-

Hydrolysis of dTTP, ATP, and GTP by RNA helicase
Assays were carried out as described under "Materials and Methods" with varying amounts of RNA helicase in the presence or absence of poly(A) as indicated. After 60 min at 30 "C, aliquots were loaded onto polyethyleneimine-cellulose plates, which were developed and quantitated as described in the text.   (27). RNA helicase I also differs from the human p68 RNA helicase since the monoclonal antibody PAb204, which cross-reacts with p68 (5), failed to immunoprecipitate the activity of the SG2 fraction and did not react with this preparation using immunoblotting procedures (data not shown).
As reported for eIF-4A plus eIF-4B (4) and the PPV CI protein (7), the displacement of femtomole levels of dsRNA required the addition of picomole quantities of protein. In contrast, femtomole levels of p68 and RNA helicase I displaced femtomole levels of dsRNA. In the case of RNA helicase I, 50 fmol of enzyme displaced 10 fmol of dsRNA, after which time the reaction came to a halt. It appeared that this effect was due to the loss of RNA helicase activity since the addition of more enzyme rather than more dsRNA resulted in the resumption of s R N A formation. The reasons for these observations are not clear. There is the possibility that the helicase is sequestered by ssRNA, which prevents reannealing of the s R N A products. We think this is unlikely because the E. coli SSB and the HSSB had little effect on the RNA helicase I activity. In addition, we did not detect significant reannealing when denatured substrates were incubated under the standard RNA helicase assay conditions in the absence of the enzyme. In the case of eIF-4A (plus eIF-4B), it has been suggested that the requirement for a large molar excess of the proteins may overcome the dissociation of the eIF-4A and eIF-4B complex, which is the active RNA helicase (4). However, such an explanation is unlikely in the case of the CI protein of PPV since its RNA helicase activity can be carried out with a single polypeptide chain. An alternative explanation is that purified helicases strongly interact with ssRNA and that additional trans-acting factor(s) may be required for efficient dissociation of the enzyme from the products.
To date, RNA helicase and RNA-dependent NTPase activities have been demonstrated for only three other proteins: mammalian translation eIF-4A (plus eIF-4B), which is thought to unwind the secondary structure of mRNA to facilitate ribosome binding (2-4); human nuclear protein p68 (5), which is thought to function in the regulation of cell growth and division (28); and the CI protein, encoded by the plum pox virus (a positive-strand ssRNA virus), which is thought to play a role in the replication of the genomic viral RNA (7). These three proteins contain seven highly conserved motifs distributed over a 300-400-amino acid domain (9, 29-33). These motifs are shared by a large number of genes from prokaryotes to man that define a superfamily of putative helicases that also includes a number of well-defined RNA and DNA helicases (32,33). Thus, it is tempting to speculate that all genes containing this domain may in fact be helicases. Interestingly, four DEAD box genes, prp5 (15), ~r p 2 8 ,~ spp81, and d b~l ,~ are involved in yeast pre-mRNA splicing. In addition, a new family of RNA helicase-like genes, the DEAH family, includes three yeast pre-mRNA splicing genes, prp2 (34), prpl6 (16), and ~r p 2 . Z~ The ubiquitous distribution of these highly conserved sequences indicates that RNA helicases may play an important role in the regulation of gene expression. Thus, the study of these enzymes may contribute J. Beggs, personal communication. to our understanding of the various steps involved in the increasing number of RNA transactions.
Another approach that has been used to identify protein members of the helicase family has been carried out in yeast by Chang et al. (31). Exploiting the DEAD box sequence and the conserved motif H-R-I-G-R, they have detected, using the polymerase chain reaction, a number of different sequences between these two conserved regions (-650 nucleotides). We have carried out experiments using a HeLa cDNA library with similar results. As in yeast, a number of individual clones were isolated that have different sequences. Some of these clones have been sequenced, and we have identified one that is highly homologous to the mouse eIF-4A protein and another that has some homology to the translation initiation factor genes of yeast.
We plan to isolate the different RNA helicases of HeLa cells and to obtain enough protein sequence data to permit the isolation of specific cDNAs coding for the helicases. We may also be able to relate these proteins to the cloned polymerase chain reaction products isolated from yeast and HeLa DNAs. It will be interesting to test whether extracts prepared from mutants in the yeast system (belonging to the RNA helicase family) can be complemented by the RNA helicases isolated from nuclear extracts of HeLa cells.