From the Department of Oncology, Cross Cancer Institute and
University of Alberta, 11560 University Ave., Edmonton,
Alberta T6G1Z2, Canada
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INTRODUCTION |
DEAD box proteins are a family of putative RNA helicases that are
characterized by eight conserved amino acid motifs, one of which is the
ATP hydrolysis motif containing the core amino acid sequence DEAD
(Asp-Glu-Ala-Asp) (1-3). Over 40 members of the DEAD box family have
been isolated from a variety of organisms including bacteria, yeast,
insects, amphibians, mammals, and plants. The prototypic DEAD box
protein is the translation initiation factor, eukaryotic initiation
factor 4A, which, when combined with eukaryotic initiation factor 4B,
unwinds double-stranded RNA (4). Other DEAD box proteins, such as p68,
Vasa, and An3, can effectively and independently destabilize/unwind
short RNA duplexes in vitro (5-7). Although some DEAD box
proteins play general roles in cellular processes such as translation
initiation (eukaryotic initiation factor 4A (4)), RNA splicing (PRP5, PRP28, and SPP81 in yeast (8-10)), and ribosomal assembly (SrmB in
Escherichia coli (11)), the function of most DEAD box
proteins remains unknown. Many of the DEAD box proteins found in higher eukaryotes are tissue- or stage-specific. For example, PL10
mRNA is expressed only in the male germ line, and its product has
been proposed to have a specific role in translational regulation
during spermatogenesis (12). Vasa and ME31B are maternal proteins that may be involved in embryogenesis (13, 14). p68, found in dividing cells
(15), is believed to be required for the formation of nucleoli and may
also have a function in the regulation of cell growth and division (16,
17). Other DEAD box proteins are implicated in RNA degradation,
mRNA stability, and RNA editing (18-20).
The human DEAD box protein gene
DDX11 was
identified by differential screening of a cDNA library enriched in
transcripts present in the two RB cell lines Y79 and RB522A (21). The
longest DDX1 cDNA insert isolated from this library was
2.4 kb with an open reading frame from position 1 to 2201. All eight
conserved motifs characteristic of DEAD box proteins are found in the
predicted amino acid sequence of DDX1 as well as a region with homology to the heterogeneous nuclear ribonucleoprotein U, a protein believed to
participate in the processing of heterogeneous nuclear RNA to mRNA
(22, 23). The region of homology to heterogeneous nuclear
ribonucleoprotein U spans 128 amino acids and is located between the
first two conserved DEAD box protein motifs, 1a and 1b.
The proto-oncogene MYCN encodes a member of the MYC family
of transcription factors that bind to an E box element (CACGTG) when
dimerized with the MAX protein (24, 25). The MYCN gene is
amplified and overexpressed in approximately one-third of all NB tumors
(26, 27). Amplification of MYCN is associated with rapid
tumor progression and a poor clinical prognosis (26, 27). MYCN
overexpression is usually achieved by increasing gene copy number
rather than by up-regulating basal expression of MYCN (27, 28). Because
gene amplification involves hundreds to thousands of kilobase pairs of
contiguous DNA (29-32), it is possible that co-amplification of a gene
located in proximity to MYCN may contribute to the poor
clinical prognosis of MYCN-amplified tumors. The
DDX1 gene maps to the same chromosomal band as
MYCN, 2p24, and is located ~400 kb telomeric to the
MYCN gene (33-36). All four MYCN-amplified RB
tumor cell lines tested to date are amplified for DDX1
(21),2 while approximately
two-thirds of NB cell lines and 38-68% of NB tumors are co-amplified
for both genes (37-39). George et al. (39) found a
significant decrease in the mean disease-free survival of patients with
DDX1/MYCN-amplified NB tumors compared with
MYCN-amplified tumors. Similarly, Squire et al.
(38) observed a trend toward a worse clinical prognosis when both genes
were amplified in the tumors of NB patients. To date, there have been
no reports of a tumor amplified only for DDX1, and the role
that this gene plays in cancer formation and progression is not
known.
Because of the high rate of rearrangements in amplified DNA (31, 40),
it is unlikely that a gene located ~400 kb from the MYCN
gene will be consistently amplified as an intact unit unless its
product provides a growth advantage to the cell. Based on Southern blot
analysis, the DDX1 gene extends over more than 30 kb, and
there are no gross rearrangements of this gene in
DDX1-amplified tumors (21, 38). Furthermore, there is a good
correlation between DDX1 transcript levels and gene copy
number in the tumors analyzed to date. However, we need to show that
DDX1 protein is overexpressed in DDX1-amplified tumors if we
are to entertain the possibility that this protein plays a role in the
tumorigenic process. Here, we isolate and characterize the 5'-end of
DDX1 mRNA and extend the DDX1 cDNA
sequence by ~300 nt. We identify the predicted initiation codon of
DDX1 and generate antisera that specifically recognize DDX1 protein. We
analyze levels of DDX1 protein in both DDX1-amplified and
nonamplified RB and NB tumors and study the subcellular location of
this protein in the cell.
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MATERIALS AND METHODS |
Library Screening--
A human fetal brain cDNA library
(Stratagene) was screened using a 320-bp DNA fragment from the 5'-end
of the 2.4-kb DDX1 cDNA previously described (23).
Phagemids containing positive inserts were excised from
ZAP II
following the supplier's directions. The ends of the cDNA inserts
were sequenced using the dideoxynucleotide chain termination method
with T7 DNA polymerase (Amersham Pharmacia Biotech).
A human placenta genomic library (CLONTECH) was
screened with the 5'-end of DDX1 cDNA. Positive plaques
were purified, and the genomic DNA was analyzed using restriction
enzymes and Southern blotting. EcoRI-digested DNA fragments
from these clones were subcloned into pBluescript and digested with
exonuclease III and mung bean nuclease to obtain sequentially deleted
clones. The exon/intron map of the 5' portion of the DDX1
gene was obtained by comparing the sequence of DDX1 cDNA
with that of the genomic DNA.
Rapid Amplification of cDNA Ends (RACE)--
We used the
AmpliFINDER RACE kit (CLONTECH) to extend the
5'-end of DDX1 cDNA. Briefly, two µg of
poly(A)+ RNA isolated from RB522A was reverse transcribed
at 52 °C using either primer P1 or P3 (Fig. 1A). The RNA
template was hydrolyzed, and excess primer was removed. A
single-stranded AmpliFINDER anchor containing an EcoRI site
was ligated to the 3'-end of the cDNA using T4 RNA ligase. The
cDNA was amplified using either primer P2 or P4 (Fig.
1A) and AmpliFINDER anchor primer. RACE products were cloned
into pBluescript.
Primer Extension--
Poly(A)+ RNAs were isolated
from RB and NB cell lines as described previously (21, 38). The 21-nt
primers 5'-TTCGTTCTGGGCACCATGTGT-3' (primer P4 in Fig. 1A)
and 5'-TGGGACCTAGGGCTTCTGGAC-3' (primer P3 in Fig. 1A) were
end-labeled with [
-32P]ATP (3000 Ci/mmol; Mandel
Scientific) and T4 polynucleotide kinase. Each of the labeled primers
was annealed to 2 µg of poly(A)+ RNA at 45 °C for 90 min, and the cDNA was extended at 42 °C for 60 min using avian
myeloblastosis virus reverse transcriptase (Promega). The primer
extension products were heat-denatured and run on a 8% polyacrylamide
gel containing 7 M urea in 1× TBE buffer. A G + A
sequencing ladder served as the size standard.
S1 Nuclease Protection Assay--
The S1 nuclease protection
assay to map the transcription initiation site of DDX1 was
performed as described by Favaloro et al. (41). The DNA
probe was prepared by digesting genomic DNA spanning the upstream
region of DDX1 and exon 1 with AvaI, labeling the
ends with [
-32P]ATP (3000 Ci/mmol) and polynucleotide
kinase, and removing the label from one of the ends by digesting the
DNA with SphI (Fig. 4). The RNA samples were resuspended in
a hybridization mixture containing 80% formamide, 40 mM
PIPES, 400 mM NaCl, 1 mM EDTA, and the
heat-denatured SphI-AvaI probe labeled at the
AvaI site. The samples were incubated at 45 °C for
16 h and digested with 3000 units/ml S1 nuclease (Boehringer
Mannheim) for 60 min at 37 °C. The samples were precipitated with
ethanol; resuspended in 80% formaldehyde, TBE buffer, 0.1% bromphenol
blue, xylene cyanol; denatured at 90 °C for 2 min; and
electrophoresed in a 7 M urea, 8% polyacrylamide gel in
TBE buffer.
Northern and Southern Blot Analysis--
Poly(A)+
RNAs were isolated from RB and NB cell lines as described previously
(21, 38). Two µg of poly(A)+ RNA/lane were
electrophoresed in a 6% formaldehyde, 1.5% agarose gel in MOPS buffer
(20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) and transferred to nitrocellulose filter
in 3 M sodium chloride, 0.3 M sodium citrate.
The filters were hybridized to the following DNA probes,
32P-labeled by nick translation: (i) a 1.6-kb
EcoRI insert from DDX1 cDNA clone 1042 (21),
(ii) a 260-bp cDNA fragment spanning the 3'-end of DDX1
exon 1 as well as exons 2 and 3, (iii) a 160-bp fragment derived from
the 5'-end of DDX1 exon 1, and (iv)
-actin cDNA to
control for lane to lane variation in RNA levels. Filters were
hybridized and washed under high stringency. Southern blot analysis was
as described previously (21).
Preparation of Anti-DDX1 Antiserum--
To prepare antiserum to
the C terminus of the DDX1 protein, we inserted a 1.8-kb
EcoRI fragment from bp 848 to 2668 of
DDX1 cDNA (Fig. 1B) into
EcoRI-digested pMAL-c2 expression vector (New England
Biolabs). DH5
cells transformed with this vector were grown to
mid-log phase and induced with 0.1 mM
isopropyl-1-thio-
-D-thiogalactoside. The cells were
harvested 3-4 h postinduction and lysed by sonication. Soluble maltose
binding protein-DDX1 fusion protein was affinity-purified using amylose
resin, and the maltose-binding protein was cleaved with factor Xa. The
DDX1 protein was purified on a SDS-PAGE gel, electroeluted, and
concentrated. Approximately 100 µg of protein was injected into
rabbits at 4-6-week intervals. For the initial injection, the protein
was dispersed in complete Freund's adjuvant (Sigma), while subsequent
injections were prepared in Freund's incomplete adjuvant. Blood was
collected from each rabbit 10 days after injection, and the specificity
of the antiserum was tested using cell extracts from RB522A. To prepare
antiserum to the N terminus of DDX1 protein, a DDX1 cDNA
fragment from bp 268 to 851 (Fig. 1B) was inserted into
pGEX-4T2 (Amersham Pharmacia Biotech). The recombinant protein produced
from this construct contains the first 186 amino acids of the predicted
DDX1 sequence. Soluble glutathione S-transferase-DDX1 fusion
protein was purified with glutathione-Sepharose 4B (Amersham Pharmacia
Biotech). The glutathione S-transferase component of the
fusion protein was cleaved with thrombin.
Subcellular Fractionations and Western Blot Analysis--
We
used two different procedures for subcellular fractionations. First, we
isolated nuclear and S100 (soluble cytoplasmic) fractions from RB522A,
IMR-32, Y79, RB(E)-2, HeLa, and HL60 using the procedure of Dignam
(42). On average, we obtained 5-6 times more protein in the cytosolic
fractions than in the nuclear fractions. Second, 108 RB522A
cells were lysed and fractionated into S4 (soluble cytoplasmic components), P2 (heavy mitochondria, plasma membrane fragments), P3
(mitochondria, lysozymes, peroxisomes, and Golgi membranes), and P4
fractions (membrane vesicles from rough and smooth endoplasmic reticulum, Golgi, and plasma membrane) by differential centrifugation (43). We obtained 8 mg of protein in the S4 fraction, 1 mg in P2, 0.5 mg in P3, and 2 mg in P4 fraction. The procedures related to the
immunoelectron microscopy have been previously described (44).
For Western blot analysis, proteins were electrophoresed in
polyacrylamide-SDS gels and electroblotted onto nitrocellulose using
the standard protocol for protein transfer described by Schleicher and
Schuell. The filters were incubated with a 1:5000 dilution of DDX1
antiserum, a 1:200 dilution of anti-MYCN monoclonal antibody
(Boehringer Mannheim), or a 1:200 dilution of anti-actin (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). For the colorimetric analysis,
antigen-antibody interactions were visualized using either alkaline
phosphatase-linked goat anti-rabbit IgG (for DDX1) or goat anti-mouse
IgG (for MYCN) at a 1:3000 dilution. For the ECL Western blotting
analysis (Amersham Pharmacia Biotech), we used a 1:100,000 dilution of
peroxidase-linked secondary anti-rabbit IgG antibody (for DDX1) or
secondary anti-goat IgG antibody (Jackson ImmunoResearch
Laboratories).
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RESULTS |
Identification of the 5'-End of the DDX1 Transcript--
We have
previously reported the sequence of DDX1 cDNA isolated
from an RB cDNA library (21, 23). This 2.4-kb DDX1
cDNA contains an open reading frame spanning positions 1-2201 with a methionine encoded by the first three nucleotides (Fig.
1A). There is a
polyadenylation signal and poly(A) tail in the 3'-untranslated region,
indicating that the sequence is complete at the 3'-end. Manohar
et al. (37) have also isolated DDX1 cDNA from
the NB cell line LA-N-5. Their cDNA extended the 5'-end of our
sequence by 42 bp and included an additional in frame methionine
(double underlined in Fig. 1A). The possibility
of additional in frame methionines located further upstream could not
be excluded, because there were no predicted stop codons in the
upstream region of the cDNA.

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Fig. 1.
Partial sequence and structure of DDX1
cDNA. A, the sequence of the 5'-end of
DDX1 cDNA. The sequence in boldface
type starting at the asterisk was obtained using
the RACE strategy. The additional 6 bp in italic boldface
type at the 5'-end of the cDNA are predicted based on the
known DDX1 genomic sequence and primer extension analysis.
P1, P2, P3, and P4 are primers used in the RACE experiments (the
complementary sequence was used in each case). Primers P3 and P4 were
also used for the primer extension analysis. Three in frame methionine
codons are indicated by the double underline. An
in frame stop codon is indicated by the boldface
double underline. The three major transcription
initiation sites identified by primer extension are indicated by the
single arrows, while a minor site is represented
by the broad arrow. The predicted DDX1
transcription initiation sites obtained by RACE, S1 nuclease, and
primer extension are indicated as well as the 5'-ends of
DDX1 cDNA sequences obtained by screening cDNA
libraries. The sequences transcribed from exons 1, 2, and 3 are also
shown. B, the structure of the 2711-bp DDX1
cDNA is shown with an open reading frame from position 295 to
2515.
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Northern blot analysis indicated a DDX1 transcript size of
~2800 nt, suggesting that the DDX1 cDNAs isolated to
date were lacking ~300-350 bp of 5' sequence. We have used different
approaches to identify the transcription start site of DDX1.
First, we exhaustively screened a commercial fetal brain cDNA
library with the 5'-end of DDX1 cDNA. Although numerous
clones were analyzed, only one extended the sequence (by 35 bp) beyond
that published by Manohar et al. (37) (Fig.
1A).
We next used the RACE procedure in an attempt to isolate additional 5'
sequence. The nested primers used to amplify the 5'-end of the
DDX1 transcript are labeled as primers P1 and P2 in Fig. 1A and are located downstream of the three in frame
methionines (double underlined in Fig. 1A).
Poly(A)+ RNA from RB522A was reverse transcribed at
52 °C using primer P1, and the reverse transcribed cDNA was
amplified using the nested primer P2 and the 5'-RACE primer. Using this
approach, we generated a product that was 230 bp longer than any of the
cDNAs obtained by screening libraries (Fig. 1A).
Sequencing of this 230-bp cDNA revealed an in frame stop codon
(boldface double underline in Fig. 1A) located
123 bp upstream of the predicted translation initiation site. We then
prepared primers P3 and P4, located near the 5'-end of the RACE
cDNA (Fig. 1A) and repeated the RACE procedure to see if
additional 5' sequences could be obtained. The resulting RACE products
did not extend the DDX1 cDNA sequence further.
The location of the DDX1 transcription initiation site was
verified by primer extension. Poly(A)+ RNA was prepared
from the following two cell lines: DDX1-amplified RB cell
line RB522A and a nonamplified RB cell line RB(E)-2. RB522A has
elevated levels of DDX1 mRNA, while RB(E)-2 has at least
20-fold lower levels of this transcript. Three products of 40, 43, and 46 nt (with a weak signal at 45 nt) were detected in RB522A using primer P4 (Figs. 1A and 2).
The 40-nt product corresponded exactly with the 5'-end of the
RACE-derived cDNA while the 43- and 46-nt products extended the
predicted size of the DDX1 transcript by 3 and 6 nt,
respectively. None of these products were observed in RB(E)-2. Bands of
identical sizes to those obtained with RB522A mRNA were also
observed in the DDX1-amplified NB cell line BE(2)-C but not
in the DDX1-amplified NB cell line IMR-32 (data not shown). The same predicted DDX1 transcription initiation site was
identified with primer P3 except that the bands were of weaker
intensity (data not shown). We have designated the transcription start
site identified by primer extension as +1 (Fig. 1A).

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Fig. 2.
Identification of the 5'-end of the
DDX1 transcript by primer extension. Radioactively
labeled primer P4 was annealed to 2 µg of poly(A)+ RNA
from RB522A (lane 1), 1 µg of
poly(A)+ RNA from RB522A (lane 2),
and 2 µg of poly(A)+ RNA from RB(E)-2 cells
(lane 3), and extended using reverse
transcriptase. The products were run on an 8% denaturing
polyacrylamide gel with a G + A sequencing ladder as size marker. The
primer extension products are indicated on the left. The
sizes of the products (in nt) are presented as the distance from primer
P4.
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The sequence of the 6 nt extending beyond the RACE cDNA was
obtained by comparison of the cDNA sequence with that of
DDX1 genomic DNA. Bacteriophages containing DDX1
genomic DNA were isolated by screening a human placenta library with 5'
DDX1 cDNA. Eighteen kb of DNA were sequenced from two
bacteriophages with overlapping DDX1 genomic DNA. Thirteen
exons were identified within this 18-kb region (Fig.
3) corresponding to cDNA sequences
from position 1 to 1249. The 310-bp exon 1 was by far the longest of
the 13 exons sequenced, corresponding to the entire 5'-untranslated
region of DDX1 as well as the first in frame methionine. The
sequences transcribed from exons 1, 2, and 3 are indicated in Fig.
1A.

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Fig. 3.
Genomic map of the 5'-end of
DDX1. The exons are represented by the
black boxes, and distances are in kilobase pairs.
The locations of EcoRI (E) sites are
indicated.
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Knowledge of the genomic structure of DDX1 allowed us to use
the S1 protection assay, a technique that is independent of reverse transcriptase, to further define the 5'-end of the DDX1
transcript. Poly(A)+ RNAs from six
DDX1-amplified lines (RB lines: Y79 and RB522A; NB lines:
BE(2)-C, IMR-32, LA-N-1, and LA-N-5) and six nonamplified lines (RB
lines: RB(E)-2 and RB412; NB lines, GOTO, NB-1, NUB-7, and SK-N-MC)
were hybridized to a DNA probe that extended from position
745 in the
5'-flanking DDX1 DNA to position +164 in exon 1. This DNA
probe was labeled at position +164 as indicated in Fig.
4. Nonhybridized DNA was digested with S1
nuclease, and the sizes of the protected fragments were analyzed on a
denaturing polyacrylamide gel. Bands of 150-153 nt were observed in
lane 2 (RB522A), lane 5 (BE(2)-C), and lane 8 (LA-N-1) with bands of much
weaker intensity in lane 7 (IMR-32) (Fig. 4).
Specific bands were not detected in either DDX1-amplified
Y79 and LA-N-5 or the nonamplified lines. Although the sizes of the S1
protected bands in RB522A, BE(2)-C, and LA-N-1 were 5 and 11 nt shorter
than predicted based on RACE and primer extensions, respectively, there
was general agreement with all three techniques regarding the location
of the DDX1 transcription initiation site (Fig.
1A). The smaller S1 nuclease protected products could have
arisen as the result of S1 digestion of the 5'-end of the RNA:DNA
heteroduplex because of its relatively high rU:dA content (45).

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Fig. 4.
S1 nuclease mapping of the 5'-end of the
DDX1 transcript. Two µg of poly(A)+ RNA
from four RB lines (DDX1-amplified Y79 and RB522A and
nonamplified RB(E)-2 and RB412), eight NB lines
(DDX1-amplified BE(2)-C, IMR-32, LA-N-1, and LA-N-5 and
nonamplified GOTO, NB-1, NUB-7, and SK-N-MC), and tRNA as a negative
control were hybridized to a SphI-AvaI fragment
labeled at the AvaI site with [ -32P]ATP and
polynucleotide kinase. Bands of 150-153 nt are shown in
lanes 2 (RB522A), 5 (BE(2)-C), and
8 (LA-N-1) with much weaker bands in lane
7 (IMR-32). A map of the probe indicating the transcription
initiation site identified by primer extension (+1), the
labeling site (*), and exons 1 and 2, is shown at the
bottom.
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Identification of the same transcription initiation site in three
DDX1-amplified lines suggests that this represents the bona fide start site of DDX1 transcription. However, it was not
clear why this start site was either very weak or not detected in three other amplified lines. To determine whether the 5'-end of exon 1 is
transcribed in all DDX1-amplified lines, we carried out a direct analysis of the 5'-end of the DDX1 transcript by
Northern blotting. Two probes were used for this analysis: the 5' probe contained a 160-bp fragment from bp 1 to 160 (5'-half of exon 1), and
the 3' probe contained a 260-bp fragment from bp 160 to 420 (3'-half of
exon 1 as well exons 2 and 3) (Fig. 1A). With the 3' probe,
we obtained bands of similar size and intensity in four
DDX1-amplified lines (RB522A, BE(2)-C, IMR-32, and LA-N-5). Band intensity was somewhat weaker in Y79 and stronger in LA-N-1 in
comparison with the other lines (Fig. 5).
No signal was detected in the non-DDX1-amplified line RB412.
With the 5' probe, a relatively strong signal was observed in RB522A,
BE(2)-C, and LA-N-1, while a considerably weaker but readily apparent
signal was detected in Y79, IMR-32, and LA-N-5. The signal obtained
with actin indicates that, with the exception of LA-N-1, similar
amounts of RNA were loaded in each lane and that the RNA was not
degraded. These results indicate that at least a portion of the 160-bp
5'-end of exon 1 is transcribed in all DDX1-amplified
lines.

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Fig. 5.
Northern blot analysis of the 5'-end of the
DDX1 transcript. Two µg of poly(A)+ RNA
isolated from DDX1-amplified Y79, RB522A, BE(2)-C, IMR-32,
LA-N-1, and LA-N-5 and nonamplified RB412 were electrophoresed in a
1.5% agarose-formaldehyde gel. The RNA was transferred to a
nitrocellulose filter and sequentially hybridized with a 260-bp
fragment from DDX1 cDNA from bp +160 to +420 (3'-end of
exon 1 as well as exons 2 and 3) (A), a 160-bp fragment from
DDX1 cDNA from bp +1 to +160 (5'-end of exon 1)
(B), and actin cDNA (C). The DNA was labeled
with [32P]dCTP by nick translation. The blots were
hybridized and washed under high stringency.
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Based on primer extension, S1 nuclease protection assay, Northern blot
analysis and the sequencing of the RACE products, we conclude that the
DDX1 transcript is 2.7 kb with an open reading frame
spanning nucleotides 295-2515 encoding a predicted protein of 740 amino acids with an estimated molecular weight of 82.4 (Fig.
1B). An in frame stop codon is located 123 nt upstream of the predicted translation initiation site, at positions 172-174. The
first in frame methionine following the stop codon is in agreement with
the Kozak consensus sequence (46). Furthermore, the predicted start
methionine codon for human DDX1 corresponds perfectly with that of
Drosophila DDX1 (47). A stop codon is located 15 nt upstream
of the initiation codon in Drosophila DDX1.
Analysis of DDX1 Protein Levels in Neuroblastoma and
Retinoblastoma--
We and others have previously shown that there is
a good correlation between gene copy number and RNA levels in
DDX1-amplified RB and NB cell lines (37, 38). To determine
whether the correlation extends to DDX1 protein levels, we prepared
antiserum to two nonoverlapping recombinant DDX1 proteins. First, we
prepared a C terminus recombinant protein construct by inserting a
1.8-kb EcoRI fragment from bp 848 to 2668 (amino acids
185-740) (Fig. 1B) into the pMAL-c2 expression vector.
Recombinant protein expression was induced with
isopropyl-1-thio-
-D-thiogalactoside, and the 110-kDa
maltose-binding protein-DDX1 fusion product was purified by affinity
chromatography using amylose resin, followed by electrophoresis on a
SDS-PAGE gel after cleaving the maltose-binding protein fusion partner
with factor Xa. Second, we prepared an N terminus construct by ligating
a DNA fragment from bp 268 to 851 (amino acids 1-186) into pGEX-4T2.
The 50-kDa glutathione S-transferase-DDX1 fusion protein was
purified by affinity chromatography on a glutathione column. This N
terminus fusion protein contains only the first of the eight conserved
motifs found in all DEAD box proteins, while the C terminus fusion
protein includes the remaining seven motifs.
We measured DDX1 protein levels in total cell extracts of three RB and
10 NB cell lines. Using antiserum to the N terminus fusion protein, we
observed a strong signal in all DDX1-amplified cell lines:
the RB cell lines Y79 (lane 1) and RB522A
(lane 2) and the NB cell lines BE(2)-C (lane
4), IMR-32 (lane 6), LA-N-1 (lane 8), and LA-N-5 (lane
9) (Fig. 6). Two bands were
observed in the majority of extracts. Of the amplified lines, Y79
produced the weakest signal, with the most intense signal observed in
LA-N-1. There was an excellent correlation with DDX1 protein and
mRNA levels in these cell lines, with lower levels of DDX1 mRNA
observed in Y79 and higher levels in LA-N-1 (Fig.
7A). As shown in Fig. 7B, this correlation extended to DDX1 gene copy
number. No gross DNA rearrangements were seen in the
DDX1-amplified lines; however, three small bands of altered
size were observed in the RB412 lane. Although the nature of the DNA
alteration is not known, it is noteworthy that DDX1
transcript levels in RB412 are extremely low (Fig. 7A) and
that the top DDX1 protein band in RB412 cell extracts is smaller in
size than the top band from the other cell extracts (Fig. 6).

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Fig. 6.
DDX1 protein expression in RB and NB cell
lines. Western blots were prepared using total cellular extracts
from three RB (Y79, RB522A, and RB412) and 10 NB cell lines (BE(2)-C,
GOTO, IMR-32, KAN, LA-N-1, LA-N-5, NB-1, NUB-7, SK-N-MC, and SK-N-SH).
The lines that are amplified for the DDX1 gene are Y79,
RB522A, BE(2)-C, IMR-32, LA-N-1, and LA-N-5. Twenty µg of protein
were loaded in each lane and electrophoresed in a 10% SDS-PAGE gel.
DDX1 was detected using a 1:5000 dilution of the antiserum to the amino
terminus of DDX1 protein. Size markers in kilodaltons are indicated on
the side.
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Fig. 7.
Northern and Southern blot analyses of
DDX1 in RB and NB cell lines. A, 2 µg of
poly(A)+ RNA were loaded in each lane, electrophoresed in a
1.5% agarose-formaldehyde gel, and transferred to a nitrocellulose
filter. The filter was first hybridized to a 32P-labeled
1.6-kb DDX1 cDNA (clone 1042) (21), stripped, and rehybridized to
actin DNA. B, 10 µg of genomic DNA from each of the
indicated cell lines were digested with EcoRI,
electrophoresed in a 1% agarose gel, and transferred to a
nitrocellulose filter. The filter was hybridized to
32P-labeled clone 1042 DDX1 cDNA, stripped,
and reprobed with labeled -fetoprotein cDNA. Markers (in
kilobase pairs) are indicated on the side.
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Two DDX1 protein bands were present in most of the lanes in Fig. 6. The
same two bands were detected with antiserum to the C terminus of the
DDX1 protein, as well as a third band at ~60 kDa (data not shown).
There was no variation in the intensity of the 60-kDa band in
DDX1-amplified and nonamplified cell extracts. The 60-kDa
band probably represents another member of the DEAD box protein family,
because the C terminus DDX1 protein used to prepare this antiserum
contained seven of the eight conserved motifs found in all DEAD box
proteins. To obtain an estimate of the size of the two DDX1 bands, we
ran cellular extracts from RB522A on a 7% SDS-PAGE gel with the
BenchMark protein ladder (Life Technologies, Inc.). The size of the
DDX1 protein was determined using the Alpha Imager 2000 documentation
and analysis system for molecular weight calculation. Based on this
analysis, the estimated molecular mass of the top band is 89.5 kDa,
while that of the bottom band is 83.5 kDa. The 84-kDa band may
represent the unmodified product encoded by the DDX1 transcript
(capable of encoding a protein with a predicted molecular mass of 82.4 kDa), while the top band may represent post-translational modification of DDX1 protein (e.g. phosphorylation). Another possibility
is that the top band represents intact DDX1 and the lower band is a
specific truncated or degradation product of DDX1. Yet a third possibility is that the two bands represent the products of
differentially spliced transcripts or different translation initiation
sites. However, the lack of any obvious differences in DDX1
transcript sizes in the three RB and 10 NB lines analyzed in Fig.
7A does not support the latter possibility (e.g.
compare the DDX1 transcript size in NUB-7 (which produces
the lower DDX1 protein band) and in NB-1 (which produces the higher
DDX1 protein band)).
Subcellular Localization of DDX1 Protein--
DEAD box proteins
have been implicated in a variety of cellular functions including RNA
splicing in the nucleus, translation initiation in the cytoplasm, and
ribosome assembly in the nucleolus. To obtain an indication of the
possible role of DDX1, we studied its subcellular location. Nuclear and
cytosolic extracts were prepared from DDX1-amplified RB522A
and run on a 7% SDS-PAGE gel. Although there was more DDX1 protein in
the cytosol than in the nucleus on a per cell basis, the proportion of
DDX1 protein relative to total protein was similar in both cellular
compartments (Fig. 8A). Both
the 90- and 84-kDa bands were present in cytosol and nuclear extracts,
although the bottom band was more readily apparent in the cytosol. By
running the gel for an extended period of time (twice as long as
usual), we were able to detect an additional weak band at ~88 kDa in
both nuclear and cytosolic extracts.

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Fig. 8.
Distribution of DDX1 in the nucleus and
cytoplasm. A, cytosolic and nuclear extracts were
prepared from RB522A and electrophoresed in a 7% SDS-PAGE gel.
Cytosolic extracts were loaded in lanes 1 (20 µg of protein) and 2 (10 µg), while nuclear extracts
were loaded in lanes 3 (10 µg) and 4 (20 µg). DDX1 was visualized using a 1:5000 dilution of the antiserum
to the N terminus. The BenchMark protein ladder size markers
(kilodaltons) are indicated on the left. B,
cytosolic and nuclear extracts were prepared from HL60, Y79, IMR-32,
HeLa, RB522A, and RB(E)-2 and electrophoresed in an 8% SDS-PAGE gel.
Twenty µg of proteins were loaded in each lane marked
C (cytosolic) and N (nuclear). DDX1 was
visualized using a 1:5000 dilution of the antiserum to the N terminus.
Actin levels were analyzed using a 1:200 dilution of anti-actin
antibody (Santa Cruz Biotechnology).
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To determine whether DDX1 consistently localizes to both the cytoplasm
and nucleus, we prepared cytosol and nuclear extracts from two
additional DDX1-amplified lines, Y79 and IMR-32, as well as
from nonamplified RB(E)-2, HL60, and HeLa. DDX1 protein was found in
both the nucleus and cytoplasm of IMR-32, primarily in the cytoplasm of
Y79, and mainly in the nucleus of the three nonamplified lines (Fig.
8B). In addition, DDX1 was almost exclusively found in
nuclear extracts prepared from normal GM38 fibroblasts (data not
shown). We used anti-actin antibody to ensure that our nuclear and
cytosolic extracts were not cross-contaminated (Fig.
8B).
We next carried out a more detailed analysis of DDX1 subcellular
location using two different approaches: (i) fractionation of cellular
components into nuclei; S100 or S4 cytosol (containing soluble
cytoplasmic components, including 40 S ribosomes); P2 (heavy
mitochondria, plasma membrane fragments plus material trapped by these
membranes); P3 (mitochondria, lysosomes, peroxisomes, Golgi membranes,
some rough endoplasmic reticulum); and P4 (microsomes from smooth and
rough endoplasmic reticulum, Golgi and plasma membranes) (43); and (ii)
immunogold electron microscopy. The DDX1-amplified RB522A
cell line was used for both experiments. The fractionation procedures
indicate that DDX1 is mainly in the nucleus and in the cytosol (S4 and
S100 fractions) of RB522A cells (Fig.
9A). As a control, we used
anti-human MYCN antibody to determine the location of MYCN (also
amplified in RB522A) in our subcellular fractions. As shown in Fig.
9B, MYCN was primarily found in the nucleus, as one would
expect of a transcription factor.

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Fig. 9.
Subcellular location of DDX1 protein.
RB522A cells were fractionated into nuclear (lane
1), S100 and S4 cytosol (lanes 2 and
3), P2 membrane (lane 4), P3 membrane
(lane 5), and P4 membrane (lane
6) fractions. Twenty µg of protein were loaded in each
lane and run on a 10% SDS-PAGE gel. A, DDX1 protein was
detected using a 1:5000 dilution of the antiserum to the N terminus of
DDX1. B, MYCN protein was detected using a commercially
available antibody at a 1:200 dilution. Size markers (kilodaltons) are
indicated on the side.
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For the electron microscopy analysis, antiserum to the N terminus of
DDX1 was coupled to protein A gold particles, and the distribution of
DDX1 was examined in RB522A cells fixed in paraformaldehyde and
glutaraldehyde. DDX1 was present in both the cytoplasm and nucleus
(data not shown). There was no association with either cell organelles
or with nuclear or plasma membranes.
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DISCUSSION |
There are presently few clues as to the function of DDX1 in normal
and cancer cells. Our earlier data indicate that DDX1
mRNA is present at higher levels in fetal tissues of neural origin (retina and brain) compared with other fetal tissues (21). There may
therefore be a requirement for elevated levels of this putative RNA
helicase for the efficient production or processing of neural specific
transcripts. A role in cancer formation or progression is an intriguing
possibility, because overexpression of an RNA unwinding protein could
affect the secondary structure of RNAs in such a way as to alter the
expression of specific proteins in tumor cells. DDX1 is
co-amplified with MYCN in a subset of RB and NB cell lines
and tumors (37-39). MYCN amplification is common in stage
IV NB tumors and is a well documented indicator of poor prognosis. A
general trend toward a poorer clinical prognosis is observed when both
the MYCN and DDX1 genes are amplified compared with when only MYCN is amplified (38, 39), suggesting a
possible role for DDX1 in NB tumor formation or progression.
It is generally accepted that co-amplified genes are not overexpressed
unless they provide a selective growth advantage to the cell (48, 49).
For example, although ERBA is closely linked to
ERBB2 in breast cancer and both genes are commonly amplified in these tumors, ERBA is not overexpressed (48). Similarly, three genes mapping to 12q13-14 (CDK4, SAS, and
MDM2) are overexpressed in a high percentage of malignant
gliomas showing amplification of this chromosomal region, while other
genes mapping to this region (GADD153, GLI, and
A2MR) are rarely overexpressed in gene-amplified malignant
gliomas (50, 51). The first three genes are probably the main targets
of the amplification process, while the latter three genes are probably
incidentally included in the amplicons. The data shown here indicate
that DDX1 is overexpressed at both the protein and RNA levels in
DDX1-amplified RB and NB cell lines and that there is a
strong correlation between DDX1 gene copy number,
DDX1 RNA levels, and DDX1 protein levels in these lines. Our
results are therefore consistent with DDX1 overexpression playing a
positive role in some aspect of NB and RB tumor formation or
progression. Recently, Weiss et al. (52) have shown that transgenic mice that overexpress MYCN develop NB tumors several months
after birth. They conclude that MYCN overexpression can contribute to
the initiation of tumorigenesis but that additional events are required
for tumor formation. Amplification of DDX1 may represent one
of many alternative pathways by which a normal precursor
"neuroblast" or "retinoblast" cell gains malignant
properties.
The function of the majority of tissue-specific or developmentally
regulated DEAD box genes remains unknown. However, some members of this
protein family have been either directly or indirectly implicated in
tumorigenesis. For example, the p68 gene has been found to be mutated
in the ultraviolet light-induced murine tumor 8101 (53), while DDX6
(also known as RCK or p54) is encoded by a gene located at the
breakpoint of the translocation involving chromosomes 11 and 14 in a
cell line derived from a B-cell lymphoma (54, 55). Similarly, the
production of a chimeric protein between DDX10 and the
nucleoporin gene NUP98 has been proposed to be involved in
the pathogenesis of a subset of myeloid malignancies with inv(11)
(p15q22) (56). Interestingly, Grandori et al. (57) have
shown that MYCC interacts with a DEAD box gene called MrDb, suggesting that the transcription of some DEAD box genes could be
regulated through interaction with members of the MYC family. Future
work will involve determining whether DDX1 represents another member of
the DEAD box family with a role in the tumorigenic process.
DEAD box proteins have been implicated in translation initiation, RNA
splicing, RNA degradation, and RNA stability (3, 18, 19). We carried
out subcellular localization studies in an attempt to obtain a general
indication of the function of DDX1. We found DDX1 protein in both the
cytoplasm and nucleus of DDX1-amplified NB and RB lines. In
contrast, DDX1 was mainly located in the nucleus of nonamplified cell
lines and normal fibroblast cultures. DDX1 was not associated with
cellular organelles or with membranes based on immunoelectron
microscopy. We therefore propose that the primary role of DDX1 is in
the nucleus. The presence of DDX1 in the cytoplasm of
DDX1-amplified cells may indicate that the amount of DDX1
protein that is allowed in the nucleus is tightly regulated.
Alternatively, DDX1 may play a dual role in the nucleus and cytoplasm
of DDX1-amplified cells.
An important component of our analysis was to identify the translation
and transcription initiation sites of DDX1. We used a
combination of techniques to identify the transcription start site:
screening of RB and fetal brain libraries, RACE, primer extension,
genomic DNA sequencing, S1 nuclease mapping, and Northern blot analysis
using probes to the predicted 5'-end of the transcript. The
transcription start site identified using these techniques is located
~300 nt upstream of the predicted translation initiation codon and
was readily detected in three DDX1-amplified lines and barely detectable in a fourth amplified line. The 5'-untranslated region as well as the first in frame methionine are encoded within the
first exon of DDX1. An in frame stop codon is located 123 nt
upstream of the predicted initiation codon. We were unable to identify
the transcription initiation site of DDX1 in two of the six
amplified lines tested as well as in nonamplified lines. Although it
remains possible that there are different transcription start sites in
different cell lines, detection of lower levels (rather than the
absence) of the 5'-most 160 nt of the DDX1 transcript in
IMR-32, Y79, and LA-N-5 compared with RB522A, BE(2)-C, and LA-N-1
supports a quantitative rather than a qualitative difference in the
5'-end of this transcript in these cells. Our results suggest that the
5'-end of DDX1 mRNA is rarely intact, even in mRNA
preparations that otherwise appear to be of high quality based on
analysis of control transcripts. The 5'-end of DDX1 mRNA
may therefore be especially susceptible to degradation, perhaps because
of its sequence and/or secondary structure.
In conclusion, we have mapped the 5'-end of the 2.7-kb DDX1
transcript and have identified the predicted translation initiation site of DDX1 protein. We have found that DDX1-amplified RB
and NB tumor lines overexpress DDX1 protein and that there is a good correlation between gene copy number and both transcript and protein levels in these cells. We have shown that DDX1 protein is primarily located in the nucleus of cells that are not DDX1-amplified.
In contrast, DDX1 is present in both the nucleus and cytoplasm of DDX1-amplified NB and RB lines. A cytoplasmic location in
DDX1-amplified lines may indicate that the amount of nuclear
DDX1 is tightly regulated or that DDX1 plays a dual role in the
cytoplasm and nucleus of these cells.
We thank Walter Dixon, Brenda Gallie, Ajay
Pandita, Jeremy Squire, and Herman Yeger for the neuroblastoma and
retinoblastoma cell lines. We thank Halyna Marusyk for carrying out the
electron microscopy analyses. We are grateful to Randy Andison for
expert help in the preparation of the manuscript and to Stacey Hume for helpful discussions.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X70649.