Download Intracellular localization of NDH II - Journal of Cell Science

1055
Journal of Cell Science 112, 1055-1064 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0230
Pre-mRNA and mRNA binding of human nuclear DNA helicase II (RNA
helicase A)
Suisheng Zhang1, Christine Herrmann2 and Frank Grosse1,*
1Department
of Biochemistry and 2Department of Electron Microscopy and Molecular Cytology, Institute for Molecular
Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 26 January; published on WWW 10 March 1999
SUMMARY
Nuclear DNA helicase II (NDH II), alternatively named RNA
helicase A, seems to function as a pre-mRNA and mRNA
binding protein in human cells. Immunofluorescence studies
of NDH II gave a highly diffused nucleoplasmic staining that
was similar to that of hnRNP A1 but differed from the
localization of the RNA splicing factor Sc-35. Upon
transcriptional inhibition, NDH II migrated from the
nucleus into the cytoplasm. During mitosis, NDH II was
released into the cytoplasm during pro- to metaphase, and
was gradually recruited back into telophase nuclei. The
timing of nuclear import of NDH II at telophase was found
to be later than that of hnRNP A1 but paralleled that of Sc35. At the ultrastructural level, both NDH II and hnRNP A1
were identified within perichromatin ribonucleoparticle
fibrils. However, the subnuclear distributions of NDH II and
hnRNP A1 were not overlapping. NDH II could be extracted
together with poly(A)-containing mRNA from HeLa cell
INTRODUCTION
Nuclear DNA helicase II (NDH II) was originally isolated from
calf thymus as an unwinding enzyme thought to be involved in
DNA metabolism (Zhang and Grosse, 1991). Subsequently it
was shown that NDH II is able to unwind both DNA and RNA,
which brought about the possibility that this enzyme may also
be involved in RNA synthesis (Zhang and Grosse, 1994). A
function in RNA rather than DNA metabolism was further
strengthened by identifying the cDNA sequence of bovine NDH
II (Zhang et al., 1995). The sequence revealed that NDH II is a
close homologue of human RNA helicase A (RHA) (Lee and
Hurwitz, 1992) and the Drosophila ‘maleless’ protein (MLE)
(Kuroda et al., 1991), displaying 92% and 80% amino acid
similarities, respectively. Only recently it was demonstrated that
RHA and MLE also unwind both RNA and DNA, providing
evidence that this group of enzymes shares identical biochemical
properties (Lee et al., 1997). The three NDH II homologues
catalyze nucleic acid unwinding that is fueled by the hydrolysis
of any of the four NTPs or dNTPs; the KM value for ATP is only
≈10 µM. All these enzymes need a 3′-single stranded tail as entry
site for nucleic acid displacement and migrate from 3′ to 5′ with
nuclei and, to a much lesser extent, from the cytoplasm.
Following transcriptional inhibition, NDH II was
preferentially associated with mRNA from the cytosol, which
biochemically confirmed the microscopic observations.
Although NDH II is mainly a nuclear enzyme, it is
apparently not associated with the nuclear matrix, since it
could be extracted with 2 M NaCl from DNase I-treated
nuclei. Our cellular and biochemical observations strongly
suggest that NDH II is a pre-mRNA and mRNA binding
protein. Its significant affinity for ssDNA, but not for dsDNA,
points to a transient role in DNA binding during the process
of transcript formation. According to our model, singlestranded DNA might be necessary to retain NDH II in the
nuclear compartment.
Key words: DEXH family, Immunoelectron microscopy,
Immunofluorescence, Mitosis, MLE, hnRNP A1, poly(A) RNA, Sc-35
respect to the single-strand they have bound to (Lee et al., 1997;
Lee and Hurwitz, 1992; Zhang and Grosse, 1991). The
Drosophila MLE protein is involved in sex-specific gene dosage
compensation by increasing the transcriptional level of the single
X-chromosome of males to reach that of the two female Xchromosomes (Kuroda et al., 1991; Lee et al., 1997). This is
obviously achieved by binding of MLE to the only X
chromosome of males, whereas it does not decorate the two Xchromosomes of female flies. Specific labeling of the single X
chromosome could be destroyed by RNase treatment, pointing
to a preferred binding of RNA structures (Richter et al., 1996).
Moreover, recently it has been shown that RHA/NDH II binds
to the constitutive cytoplasmic transport element of some
retroviral genomic RNAs, indicating both recognition of RNA
structures and a putative function in nucleocytoplasmic transport
(Tang et al., 1997). Also, RHA/NDH II was shown to be required
as ‘bridging element’ for the attraction of the transcriptional
coactivator CBP/p300 to the cellular RNA polymerase II
complex (Nakajima et al., 1997). All these findings strongly
suggest a role of NDH II and its homologues in RNA rather than
DNA metabolism, despite the fact that NDH II was originally
identified as DNA unwinding enzyme.
1056 S. Zhang, C. Herrmann and F. Grosse
To further characterize the cellular function of NDH II we
analyzed its intracellular distribution by immunofluorescence
and immunoelectron microscopy. Our immunofluorescence
studies revealed a similar cellular distribution pattern as the
mRNA packaging factor hnRNP A1 and a different localization
than the RNA splicing factor Sc-35. Observations by
immunoelectron microscopy revealed a largely nonoverlapping association of NDH II and hnRNP A1 with
perichromatin RNP fibrils. Biochemically, we could show that
NDH II copurifies with poly(A)-containing RNAs.
Nevertheless, purified NDH II binds equally well to singlestranded RNA (ssRNA), double-stranded RNA (dsRNA), and
with only a slightly decreased affinity to single-stranded DNA
(ssDNA), while double-stranded DNA (dsDNA) is obviously
not a substrate. From the combined cell biological and
biochemical data we suggest a model, in which NDH II
initially binds to ssDNA provided by the transcriptional
complex, then moves to the nascent transcript, unwinds part of
it to allow splicing and transport, and then returns to singlestranded parts of chromatin DNA in order to start a new cycle
of pre-mRNA association.
MATERIALS AND METHODS
Immunofluorescence
HeLa cells, grown on coverslips, were rinsed with PBS (10 mM
sodium phosphate, pH 7.4, 140 mM NaCl, 3 mM KCl), fixed in 4%
paraformaldehyde in PBS for 15 minutes and then permeabilized in
0.5% Triton X-100 in PBS for another 15 minutes. To reduce the nonspecific background, cells were blocked by incubation with 5% bovine
serum albumin (BSA) in PBS for 15 minutes. After washing with
PBS, primary antibodies in PBS containing 0.5% BSA were added
with different titers, as indicated in the figure legends. Incubation with
the antigen-specific antibody was for 1 hour, followed by a PBS wash
and further incubation for 1 hour with Cy3-labelled anti-rabbit IgG
(Amersham, Braunschweig, Germany) at a dilution of 1 to 400 or antimouse IgG conjugated with fluorescein isothiocyanate (FITC)
(Boehringer-Mannheim, Mannheim, Germany) at a dilution of 1 to
60. Immunofluorescence was viewed with a Zeiss fluorescence
microscope and photographed under 100-fold magnification. Doubleimmunofluorescent labeling was achieved by co-incubation of cells
with rabbit and mouse primary antibodies, which were recognized by
Cy3- or FITC-coupled secondary antibodies against rabbit or mouse
IgG.
Electron microscopy
HeLa cells were fixed in 4% paraformaldehyde in 0.1 M Sörensen
phosphate buffer, pH 7.3, at 4°C for 6 hours. After washing in 0.1 M
Sörensen buffer for three times, the cell pellet was dehydrated in
ethanol with gradually increasing concentrations (from 30% to 75%)
and then embedded in LR White resin (Plano, Cardiff, UK). Ultrathin sections were treated with 5% BSA in PBS for 1 hour and
incubated for 6 hours with rabbit and mouse primary antibodies at the
same dilutions described for immunofluorescence, followed by an
overnight incubation with gold-conjugated secondary antibodies
against rabbit IgG and mouse IgG at dilutions of 1 to 100 (Plano).
After immunolabeling, samples were proceeded for the EDTA
regressive method developed for electron microscopy observations of
RNA-containing structures (Bernhard, 1969).
Purification of recombinant human NDH II
Human NDH II was overexpressed in baculovirus-infected Sf9 cells
and purified by Ni2+-NTA-agarose chromatography and subsequent
chromatography on poly(rI•rC) agarose, exactly as described (Zhang
and Grosse, 1997).
Co-purification of NDH II with poly(A)-containing RNA
from HeLa cells
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM, C.C.Pro GmbH, Neustadt/W., Germany) supplemented with
10% fetal bovine serum (Gibco-BRL, Karlsruhe, Germany). To examine
binding of NDH II to cellular RNA substrates, HeLa cells (≈4×107) were
collected, washed with PBS and suspended in 500 µl of RSB-100 buffer
(10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.2, 0.5% Triton
X-100, 0.1% Trasylol (Bayer, Leverkusen, Germany) and 1 mM
phenylmethanesulfonyl fluoride (PMSF)). The cells were disrupted by
passing the suspension through a 25-gauge needle for 4 times, followed
by centrifugation at 3000 g at 4°C for 5 minutes. The cytosolic
supernatant fraction was mixed with an equal volume of 2× oligo(dT)
column loading buffer (1×, 20 mM Tris-HCl, pH 7.8, 0.5 M NaCl, 1 mM
EDTA, 0.1% SDS and 0.1 M 2-mercaptoethanol). Separately, the nuclear
pellet was suspended in 500 µl of cold RSB-100 buffer, sonicated and
centrifuged at 12,000 g at 4°C for 15 minutes. The supernatant of the
nuclear extract was mixed with an equal volume of 2× oligo(dT) column
loading buffer. Both cytosolic and nuclear fractions were passed through
oligo(dT)-cellulose columns (200 µl), which have been pre-treated by
0.1 N NaOH followed by extensive washing in water until the pH was
7.0 and then equilibrated with 10 volumes of 1× oligo-dT column
loading buffer. After loading, the columns were washed with 20 volumes
of 1× oligo(dT) column-loading buffer. The columns were eluted with
500 µl of 10 mM Tris-HCl, pH 7.6, 1 mM EDTA and 0.05% SDS. The
eluted fractions were incubated with 0.1 mg/ml RNase A at room
temperature for 10 minutes, followed by protein precipitation in 10%
trichloroacetic acid. After centrifugation the protein precipitates were
dissolved in 50 µl of SDS-PAGE loading buffer. One-twentieth volume
of each sample was loaded for western-blot analysis.
HeLa cell fractions and preparation of the nuclear matrix
HeLa cells (≈4×107) were collected and washed with cold PBS. Cells
were disrupted with 5 strokes of a Teflon homogenizer in 1 ml buffer
A (50 mM Tris-HCl, pH 7.8, 5 mM KCl, 5 mM MgCl2, 250 mM
sucrose, 10 mM Na2S2O5, 7 mM β-mercaptoethanol, 1 mM PMSF)
followed by centrifugation at 600 g at 4°C for 10 minutes. The
supernatants were designated as cytosolic fraction. The pellets were
suspended in 1 ml of buffer A containing 150 mM KCl and purified
by centrifugation at 2000 g at 4°C for 10 minutes through a cushion
of buffer A containing 150 mM KCl and 0.7 M sucrose. The purified
nuclei were suspended in 500 µl of buffer B (20 mM Tris-HCl, pH 7.4,
5 mM MgCl2, 250 mM sucrose, 1 mM PMSF) and DNase I was added
to 1/10 of the amount of DNA. After incubation at room temperature
for 30 minutes, the DNase I-digested nuclei suspension was
centrifuged at 1000 g for 10 minutes. Then the pellet was extracted
with 2 M NaCl in buffer C (100 mM Tris-HCl, pH 7.4, 0.1 mM MgCl2,
290 mM sucrose) for 10 minutes on ice, followed by centrifugation at
15,000 g at 4°C for 10 minutes. High-salt extraction of nuclear pellets
was repeated for three times and samples were prepared from the
supernatant and pellet of each extraction for western-blot analysis.
Filter binding assays
Filter binding assays were performed as described before (Grosse et
al., 1986). Purified NDH II was incubated at different concentrations
with 32P-labeled RNA and DNA substrates in 10 µl binding buffer
containing 20 mM Hepes, pH 8.0, 50 mM NaCl, 1 mM DTT, and 1
mM EDTA. After incubation at room temperature for 20 minutes, the
samples were loaded to a nitrocellulose membrane (Protran B85, 0.45
µM, Schleicher and Schuell, Dassel, Germany) in a 96-well vacuum
blotter. After washing for three times by filling up the slots with ≈500
µl binding buffer (each), the filter was removed, dried, and cut out at
the positions of sample loading. The retained radioactivity was
quantified by scintillation counting. In order to decrease unspecific
binding, the nitrocellulose membrane was treated with 0.3 M NaOH
for 10 minutes, washed two times for 5 minutes each with distilled
water, and then equilibrated in binding buffer for at least 16 hours.
Intracellular localization of NDH II 1057
Gel mobility-shift assays
RNA probes were transcribed by T3 and T7 RNA polymerases
(Boehringer-Mannheim) starting at the corresponding promoters of the
Bluescript KS+ plasmid (Stratagene, Amsterdam, The Netherlands).
An 85 bp long ssRNA was synthesized from the T3 promotor of this
plasmid after linearization with BamHI. The transcript was annealed
to the complementary strand that was synthesized from the T7
promotor of the Pvu II-linearized plasmid. Single-stranded protrusions
were cut-off by treating the hybrid with RNases A and T1. The RNA
probe was labeled by including [α-32P]GTP into the transcription
mixture. Purified NDH II was incubated with ≈60 µM (nt) labeled
RNA in 10 µl binding buffer (see above). After incubation at room
temperature for 25 minutes, each sample was mixed with 3 µl loading
buffer containing 40% sucrose, 0.25% Bromophenol Blue, and 0.25%
Xylene cyanol FF, and then electrophoresed through a 6% native
polyacrylamide gel in 45 mM boric acid, 45 mM Tris base, 1 mM
EDTA at 100-150 V for 50 minutes. After electrophoresis the gel was
exposed to an X-ray film at –70°C for 16-24 hours. Similarly, binding
of NDH II to M13 ssDNA was measured by incubating various
concentrations of the purified enzyme with 6 µM (nt) M13mp18 DNA,
primed with the 32P-labeled 45-mer oligodeoxynucleotide containing
a 3′-tail (Zhang and Grosse, 1997). The samples were electrophoresed
through 1% agarose in 90 mM boric acid, 90 mM Tris base, 2 mM
EDTA at 2 V/cm for 24 hours.
RESULTS
Intracellular localization of NDH II
The intracellular localization of NDH II was investigated by
double-immunofluorescence of HeLa cells with rabbit
antiserum against NDH II and mouse monoclonal antibodies
against hnRNP A1 or Sc-35. The latter ones were used as
reference markers for the intracellular distribution of NDH II
under various experimental conditions. In HeLa cells NDH II
was mainly localized in the nuclei, although very weak
cytoplasmic signals could also be seen (Fig. 1A,C). The nuclear
distribution of NDH II could be observed as a widespread
homogeneous staining within the nucleoplasmic region that
excluded the nucleoli. Similar staining patterns were also found
for hnRNP A1 (Fig. 1A′). hnRNP A1 undergoes relocalization
into the cytoplasm when transcription is inhibited (Piñol-Roma
and Dreyfuss, 1992). A similar effect was also observed for
NDH II after treatment of cells with actinomycin D (Fig. 1B).
However, following transcriptional inhibition the increased
cytoplasmic signal of NDH II was less apparent than that of
hnRNP A1 (Fig. 1B′). This might either reflect different levels
of abundance of these two proteins in HeLa cells, or, different
mechanisms regulating their cellular localization.
In contrast to hnRNPs, snRNPs and non-snRNP RNA
splicing factors are concentrated in nuclear speckles, which
occupy distinct portions of the nucleoplasmic region (Spector
et al., 1991). Some DEXD/H helicases involved in RNA
splicing colocalize with Sc-35 in nuclear speckles (Gee et al.,
1997; Molnar et al., 1997; Ono et al., 1994; Sukegawa and
Blobel, 1995). Sc-35 speckles underwent dynamic changes that
correlated with the transcriptional activity of the cell. In
transcriptionally active cells Sc-35 was dispersed throughout
the nucleoplasmic regions. But following transcriptional
inhibition Sc-35 assembled into speckles (Fig. 1D′). The
nucleoplasmic immunostaining of NDH II did not show the
same pattern as the nucleoplasmic distribution of Sc-35 (Fig.
1C,C′). Moreover, in HeLa cells treated with the transcription
inhibitor
5,6-dichloro-1-β-D-ribofuranosylbenzimidazole
(DRB), NDH II concentrated at a few faintly visible nuclear
foci, whose positions did not overlap with Sc-35 (Fig. 1D).
Redistribution of NDH II in mitotic HeLa cells
During mitosis most of the proteins with functions in
transcription and RNA processing are released from the
chromosomes and transcription activities become suppressed or
ceased for the period of chromosomal condensation. NDH II was
excluded from the mitotic nucleus as early as prophase when the
chromosomes started condensing and the nuclear envelope
disrupted (Fig. 2A,B). The exclusion reached a maximum at
metaphase when the two sets of sister chromatids were highly
condensed and aligned at the metaphase plate (Fig. 2C,D). At
telophase, NDH II re-entered the nucleus at a stage when the
chromosomes in the dividing cells started to decondense and the
nuclear envelope was gradually reformed (Fig. 2E,G, and H,J).
For comparison, we observed the redistribution of hnRNP A1
and Sc-35 by double-immunolabeling in mitotic HeLa cells.
hnRNP A1 and Sc-35 were similarly excluded from prophase
and metaphase chromosomes as NDH II (data not shown).
However, hnRNP A1 was recruited earlier than NDH II into the
telophase nucleus (Fig. 2E,F), while the re-entering of Sc-35 into
telophase nuclei paralleled that of NDH II (Fig. 2H,I).
Further differences between the nuclear import of hnRNP A1,
NDH II, and Sc-35 were found by subjecting mitotic cells to
transcriptional inhibition. The nuclear import of hnRNP A1 at
telophase was sensitively blocked by actinomycin D (Fig. 3B)
or DRB (data not shown). A similar effect was observed earlier
on the transcription-dependent nuclear transport of other
hnRNP proteins (Piñol-Roma and Dreyfuss, 1991). However,
we found that actinomycin D did not block the re-import of
NDH II into telophase nuclei which otherwise excluded hnRNP
A1 (Fig. 3A). Neither was Sc-35 subjected to a similar inhibition
of nuclear import as hnRNP A1. Sc-35 re-entered the telophase
nucleus together with NDH II irrespectively of a treatment with
actinomycin D (Fig. 3D,E). DAPI stainings (see Fig. 2B,D,G,J
and Fig. 3C,F) are shown to demonstrate the state of
chromosomal condensation and cell division.
Association of NDH II with perichromatin RNP fibrils
The subnuclear localization of NDH II in HeLa cells was
studied by immunoelectron microscopy. An EDTA-regressive
staining method was used to contrast RNP-structures against
condensed chromosomal areas in the nucleus (Bernhard,
1969). NDH II, identified by 10 nm gold-particles, was found
to be associated with RNP-structures at the chromosomal
periphery in the nucleoplasm (Fig. 4A,B) that have been
designated as perichromatin fibrils. Some NDH II molecules
showed up in cytoplasmic RNP structures, implicating possible
functions of NDH II in RNA export or translation. We also
studied the distribution of hnRNP A1 in HeLa cells, which was
detected with 5 nm gold-particles. We found that hnRNP A1
was also associated with perichromatin fibrils (Fig. 4C). Some
spherical RNP particles were associated with a high density of
hnRNP A1 (indicated by arrows in Fig. 4C). An association of
hnRNP A1 with RNP particle or fibrils was also observed in
the cytoplasm (Fig. 4C). Although NDH II and hnRNP A1
displayed a similar distribution pattern on perichromatin RNP
fibrils, which might indicate an association with nascent
hnRNA transcripts, the localization of differently sized gold
1058 S. Zhang, C. Herrmann and F. Grosse
particles hardly showed a coincidence between these two
proteins (Fig. 4D). Therefore, the two pre-mRNA binding
proteins hnRNP A1 and NDH may bind to the same substrate
at different stages of processing. Alternatively, these two
proteins may have a non-overlapping substrate specificity.
NDH II is associated with poly(A)-containing RNA
from HeLa cells
Our cell biological studies indicated that NDH II is an RNAbinding protein with a preference for pre-mRNA and possibly
mRNA. To support these observations by biochemical means, we
studied whether NDH II bound to poly(A)-containing RNA.
Some RNA binding proteins of the hnRNP group (Dreyfuss et
al., 1993) and a DEXD/H RNA helicase (Ladomery et al., 1997)
Fig. 1. Immunofluorescence of NDH II in HeLa cells. The
cellular localization of NDH II was examined by
immunofluorescence with rabbit antiserum against bovine
NDH II (1:1000), and detected by a Cy3-secondary antibody
against rabbit IgG (A-D). HeLa cells were either grown under
normal culturing conditions (A and C), or treated by 5 µg/ml
actinomycin D (B), or 100 µM DRB (D) for 3.5 hours.
Double-immunofluorescence studies were performed by coincubation of NDH II antibodies with monoclonal antibodies
(1:1000) against hnRNP A1 (A′ and B′) or Sc-35 (Sigma) (C′
and D′). The latter were detected by an FITC-labeled antibody
against mouse IgG.
have been found to co-elute from oligo(dT) columns together
with the retained poly(A)-containing RNA. Analogously,
cytoplasmic and nuclear fractions of HeLa cells were loaded onto
oligo(dT) cellulose columns in the presence of 500 mM NaCl.
The columns were washed and eluted with salt-free buffer. Highsalt buffers support binding of poly(A)-containing RNA to the
oligo(dT) matrix, and disfavor protein-nucleic acid interactions,
while low-salt buffers destabilize poly(A)•oligo(dT) interactions
and stabilize protein-RNA interactions. NDH II turned out to be
a poly(A)-containing RNA-binding protein that could be
identified in both cytosolic and nuclear fractions (Fig. 5, lanes 1
and 2). Only full-length NDH II of 142 kDa was retained,
whereas degradation products, which always showed up in the
cytosol or nuclei (see Fig. 6), were not visible. Inhibition of
A
A′
B
B′
C
C′
D
D′
Intracellular localization of NDH II 1059
A
B
C
D
E
F
H
G
I
J
nuclear RNA synthesis with actinomycin D altered the
intracellular distribution of RNA-bound NDH II. As already
observed by immunofluorescence microscopy, the amount of
NDH II increased in the cytoplasm and decreased in the nuclei
A
B
C
D
E
F
Fig. 2. Immunofluorescence of NDH II in mitotic
HeLa cells. To enrich mitotic cells, 2 mM thymidine
was added to the culture for 14-16 hours to block cells
at the beginning of S phase. The thymidine-block was
removed by returning to fresh medium. Typically, after
9-10 hours, cells were progressing into mitosis.
Immunofluorescence of NDH II is shown for prophase
(A), metaphase (C), and telophase (E and H). A
double-immunofluorescence micrograph of NDH II
and hnRNP A1 (F) or Sc-35 (I) at telophase is also
shown. DAPI stainings (B, D, G and J) are presented
for visualizing the condensation state of mitotic
chromosomes.
after inhibition of RNA synthesis (Fig. 5, lanes 3 and 4). Only
very little NDH II was eluted from the oligo(dT) cellulose column
when, before loading of the column, the cellular extracts were
treated with RNase A (Fig. 5, lanes 5 and 6).
Fig. 3. Effect of actinomycin D on the recruitment of NDH II,
hnRNP A1 and Sc-35 into HeLa telophase nuclei. HeLa cells, 9
hours after the release from a thymidine block (see legend of Fig.
2), were treated with 5 µg/ml actinomycin D for 1 hour. Doubleimmunofluorescence of NDH II with hnRNP A1 (A and B) and
NDH II with Sc-35 (D and E) are shown. DAPI stainings (C and
F) are presented for visualizing telophase nuclei.
1060 S. Zhang, C. Herrmann and F. Grosse
Fig. 4. Immunoelectron microscopy of NDH II in
HeLa cells. Electron microscopic detection of
NDH II required post-embedding
immunolabeling, followed by an EDTAregressive method for contrasting RNP
structures. NDH II and hnRNP A1 can be
distinguished by two different sizes of goldparticles conjugated with the secondary antibody
against rabbit IgG (10 nm) and mouse IgG (5
nm). Results are shown for the individual
immunolabeling of NDH II (A and B), hnRNP
A1 (C), and for double-immunolabeling of NDH
II and hnRNP A1 (D). Larger arrowheads
indicate gold-particles corresponding to NDH II
(10 nm) and smaller arrowheads indicate goldparticles corresponding to hnRNP A1 (5 nm) in
nuclei or cytoplasm. The subcellular
compartments are indicated as (C) for cytoplasm
and (N) for nucleoplasm. Magnification: ×50,
000 (A,C,D); ×100,000 (B). Bars: 200 nm
(A,C,D); 100 nm (B).
Intracellular localization of NDH II 1061
Fig. 5. Co-elution of NDH II and poly(A)-containing RNA from
oligo(dT) cellulose. HeLa cytosolic and nuclear extracts were passed
through oligo(dT) cellulose (200 µl) in the presence of 500 mM
NaCl. After washings, the columns were eluted with buffer without
salt. The eluted fractions were treated with RNase A and the proteins
that co-eluted with the oligo(dT)-retained RNAs were analyzed by
western blotting with NDH II antiserum (1 to 1000 diluted).
Cytosolic and nuclear extracts were prepared from HeLa cells grown
under normal culturing conditions (lanes 1 and 2) or treated with 5
µg/ml actinomycin D for 3.5 hours (lanes 3 and 4). Only minor
amounts of NDH II co-eluted from the oligo(dT) cellulose column
after nuclear and cytosolic extracts were treated with 0.1 mg/ml
RNase A for 15 minutes at room temperature (lanes 5 and 6).
Salt-solubility of NDH II in HeLa cell nuclei
Salt-extraction provides a physical means to distinguish
between salt-soluble and salt-insoluble nuclear proteins. The
latter ones are thought to be associated with the nuclear matrix,
a proteinaceous network that might constitute the solid support
for the co-ordination of transcription and RNA processing (for
a recent review see Pederson, 1998). To examine whether NDH
II was associated with the nuclear matrix, we fractionated
HeLa cells into cytosol and nuclei. The latter were further
purified via sedimentation through a sucrose cushion of highdensity (0.7 M), followed by chromatin depletion with DNase
I and high salt extraction of the non-chromatin remnant.
western blots were performed to analyze the presence of NDH
II in different fractions of the matrix preparation. It turned out
that NDH II did not belong to the nuclear matrix proteins since
repeated extractions with 2 M NaCl depleted the HeLa nuclei
of NDH II (Fig. 6A). We studied also the high salt-solubility
of hnRNP A1 (Fig. 6B) and Sc-35 (Fig. 6C). hnRNP A1
displayed a salt-solubility similar to that of NDH II, while Sc35 was not extractable with high salt concentrations, indicating
that Sc-35 is a nuclear matrix protein.
Nucleic acids binding properties of NDH II
A salient feature of NDH II and its homologues is binding to
and unwinding of both DNA and RNA. The cell biological
studies given above as well as the previously determined
domain structure of NDH II point to a role in RNA rather than
DNA metabolism. In an approach to better understand the
substrate specificity of this enzyme, we measured the relative
affinities of NDH II for dsRNA, ssRNA, and ssDNA using
filter binding assays (Fig. 7). Binding of NDH II to poly(rI•rC)
and to poly(rI) exhibited a steep linear increase up to a plateau
level when the protein concentration was increased. In contrast,
NDH II binding to ssDNA occurred with a sigmoidal curve that
leveled off at a lower plateau than that observed with RNA.
From the binding curves, apparent association constants (KA)
were calculated as 1.6•107 M−1, 1.5•107 M−1, and 7.4•106
M−1 for binding to dsRNA, ssRNA, and ssDNA, respectively.
Hence, NDH II bound equally well to both forms of RNA, and
only by a factor of two less well to DNA.
To further discriminate between the various substrate
affinities, gel mobility shift assays were performed.
Radioactively labeled 85-mer dsRNA or M13 ssDNA were
incubated with different amounts of NDH II and subsequently
subjected to gel electrophoresis under non-denaturing
conditions. Also, nucleic acid binding of NDH II was
challenged with various amounts of poly(rI•rC), poly(rI),
poly(rC), M13 ssDNA, and M13 dsDNA in competition binding
assays. NDH II bound to both dsRNA and ssDNA in a
concentration-dependent manner (Fig. 8A,B). Binding of NDH
II to the dsRNA probe was most efficiently competed by dsRNA
and ssDNA, but apparently not by ssRNA and dsDNA (Fig.
8B). Hence, dsRNA-bound NDH II might dissociate from this
substrate when a ssDNA entry site is provided. When ssDNA
was used as binding probe, the most efficient competitor turned
out to be ssRNA rather than dsDNA (Fig. 8B).
DISCUSSION
The intracellular localization of NDH II was revealed by
immunofluorescence and immunoelectron microscopy. Both
methods confirmed the previously observed nucleoplasmic
localization (Zhang et al., 1995), but showed also the presence
of minor amounts of the protein in the cytoplasm. Moreover,
shuttling of NDH II between the nucleus and the cytoplasm has
been demonstrated by immunofluorescence after transcriptional
inhibition and during mitosis. Furthermore, NDH II was shown
to be a poly(A)-RNA binding protein that could be extracted
from nuclei under normal circumstances and from the cytosol
after transcriptional inhibition. The distribution of NDH II in
HeLa interphase cells was very similar to that of hnRNP proteins
but differed from the speckled distribution of snRNPs and
splicing factors. Until now the mechanisms that regulate the
subnuclear localization of these proteins is unclear. In interphase,
the chromosomes are decondensed and dispersed as distinct
chromosomal territories throughout the whole nucleus.
Transcription occurs on the surface of the chromosomal
domains, to which transcription factors and RNA polymerases
have easy access. Nascent RNA transcripts are packaged by
RNA-binding proteins to form ribonucleoprotein complexes,
which undergo further post-transcriptional processing and
nucleocytoplasmic transport. It has been realized that these
molecular events may be represented by fibrillar structures
1062 S. Zhang, C. Herrmann and F. Grosse
Fig. 6. Comparison of the salt-solubility of NDH
II, hnRNP A1 and Sc-35 in HeLa cell nuclei. A
nuclear-matrix preparation protocol was applied to
the fractionation of cell extracts, followed by
western blot analysis of NDH II (A), hnRNP A1
(B) and Sc-35 (C). Rabbit anti-serum against NDH
II and monoclonal antibodies against hnRNP A1
and Sc-35 (Sigma) were diluted at 1 to 1000.
Fractions were from the cytosol (C) (lane 1), nuclei
(N) (lane 2), the nuclear supernatant (NS1) and
pellet (NP1) after the first 2 M NaCl extraction of
DNase-I treated nuclei (lanes 3 and 4), the
supernatant (NS2) and pellet (NP2) after the second
2 M salt extraction (lanes 5 and 6), and the
supernatant (NS3) and pellet (NP3) after the third
salt extraction (lanes 7 and 8).
observed at the periphery of the chromosomal domains (Lamond
and Earnshaw, 1998; Misteli and Spector, 1998). hnRNPs are
associated with perichromatin fibrils and immunofluorescence
studies revealed that they are homogeneously distributed
throughout the nuclear regions excluding the nucleoli (Dreyfuss
et al., 1993). The occupation of these fibrils by both NDH II and
hnRNPs provided evidence for common functions in posttranscriptional RNA processing and transport. Indeed, NDH II
displays some similarities with hnRNP proteins by having a
modular protein structure consisting of two dsRNA binding
domains (dsRBDs) at the N terminus and an arginine/glycinerich RGG box at the C terminus (Dreyfuss et al., 1993; Zhang
and Grosse, 1997). The additional RNA binding elements may
contribute to the observed nuclear localization pattern (Dreyfuss
et al., 1993). dsRBDs are part of many nucleic acid binding
proteins, such as the components of hnRNPs, mRNPs, and
ribosomes (Bass et al., 1994; Eckmann and Jantsch, 1997). The
dsRBDs are also essential domains for some proteins involved
in RNA transport and translation (Bycroft et al., 1995; McMillan
et al., 1995). An RGG-box is not only found in NDH II but also
in many hnRNP proteins; this domain may mediate both nucleic
acid binding and protein-protein interactions (Dreyfuss et al.,
1993). Hence, it is tempting to speculate that both the dsRBDs
and the RGG-box of NDH II support binding to hnRNAs for
subsequent processing and transport.
On the other hand, a family of pre-mRNA binding proteins,
which participate in spliceosome-mediated splicing events,
contain arginine/serine (RS)-rich nucleic acid binding domains
(Kramer, 1996). RS-rich pre-mRNA binding proteins, such as
Sc-35, have a nuclear localization different from hnRNP
proteins. Although they are also found in perichromatin fibrils,
where transcription, splicing and other co-transcriptional
events occur, most of the RNA splicing factors are accumulated
in interchromatin granules, which probably represent sites for
their recycling and/or storage. Corresponding to their
distribution on perichromatin fibrils and their enrichment at
interchromatin granules, splicing factors give rise to a diffused
nucleoplasmic staining pattern and the characteristic speckled
localization. RS-domains and other nucleic acid binding
motifs, such as RRM (RNA recognition motif), seem to direct
splicing factors to these subnuclear structures, possibly via
specific protein-protein and/or protein-nucleic acid interactions
Intracellular localization of NDH II 1063
0.8
0.7
Fraction Bound
0.6
0.5
0.4
0.3
0.2
0.1
0
0
40
80
120
160
200
240
280
NDH II (nM)
poly(rI•rC) [KA = 1.6 x 107 M-1 ]
poly(rI) [KA = 1.5 x 107 M-1 ]
ssM13 [KA = 7.4 x 106 M-1 ]
Fig. 7. Quantification of NDH II binding to ssRNA, dsRNA, and
ssDNA. The indicated amounts of purified NDH II were incubated
with 32P-labeled poly(rI•rC) (open circles), poly(rI) (filled circles), or
M13 ssDNA (filled squares) in binding buffer and then passed
through nitrocellulose filters. The retained radioactivities were
determined by scintillation counting.
(Caceres et al., 1997; Li and Bingham, 1991). According to
both the subnuclear localization and primary structure, NDH
II does not belong to the group of pre-mRNA binding proteins
that functions as component of the spliceosome.
Cell biological studies revealed that NDH II most likely is
a pre-mRNA and mRNA binding protein that might be
involved in structural rearrangements of nascent transcripts for
subsequent processing and transport. This explanation,
however, did not explain the still enigmatic property of this
enzyme (and its homologues) to bind to both DNA and RNA.
This led us to measure the relative affinities of NDH II for both
nucleic acid substrates. Interestingly, NDH II bound dsRNA,
ssRNA, and ssDNA nearly equally well, but displayed a much
lower affinity for dsDNA. The low affinity for dsDNA might
explain that upon transcriptional inhibition and during mitosis,
NDH II is not retained within the nucleus. This also explains
the well-known biochemical finding that NDH II requires a
single-stranded tail as entry site for DNA unwinding (Zhang
and Grosse, 1991). Moreover, a detailed biochemical analysis
of the binding properties of the individually expressed
auxiliary binding domains of NDH II revealed that the dsRBDbound dsRNA could be competed by ssRNA and by ssDNA,
but not by dsDNA. Furthermore, the RGG box bound equally
well to ssDNA and ssRNA and again not to dsDNA (S. Zhang
and F. Grosse, unpublished results).
Thus, from combining cell biological and biochemical data
one can envision the following scenario for the action of NDH
II on pre-mRNA and mRNAs: NDH II binds equally well to
single-stranded and double-stranded regions of RNA, where
single-stranded parts may act as entry-sites for the subsequent
unwinding of dsRNA. Those unwound regions may be reannealed by hnRNPs to form correctly folded transcripts for
subsequent splicing and transport. NDH II, when in the
proximity of chromatin fibers may dissociate from re-folded
Fig. 8. Competitive gel shift assays
for studying NDH II’s relative
affinities for various nucleic acids.
(A) Recombinant and highly
purified human NDH II was
incubated with the dsRNA probe
and then subjected to gel
electrophoresis. The left panel
indicates titrations with 0-160 nM
of NDH II, and the resulting
mobility shifts. The following
panels (from left to right) indicate
competition of the mobility shift
obtained with 40 nM NDH II by the
indicated amounts of poly(rI•rC),
poly(rI), poly(rC), M13 ssDNA, and
M13 dsDNA. (B) The same
experiment as described above, but
using 6 µM (nt) M13 ssDNA as
binding substrate for NDH II. Here,
binding was competed with 1.5-15
µM (nt) poly(rI•rC) and poly(rI),
respectively.
1064 S. Zhang, C. Herrmann and F. Grosse
dsRNAs to ssDNA that is transiently formed during
transcription and thereby may facilitate a new round of
transcript formation. Alternatively, these ssDNA domains may
act as retention sites to keep NDH II within the nucleus. When
the lengths of the newly formed transcript exceeds ≈17
nucleotides, i.e. the minimal length for nucleic acid binding
(Zhang and Grosse, 1994), NDH II might use this singlestranded part of the nascent transcript as binding site to restart
the removal of randomly formed secondary structures from the
newly formed pre-mRNA. Therefore, NDH II might act as
molecular chaperone that removes unwanted hybrids between
two neighboring pre-mRNA molecules in heavily transcribed
genes. Upon transcriptional inhibition or during mitosis, NDH
II cannot be kept in the nucleus because of the lack of singlestranded nucleic acids. Hence, it migrates together with mRNA
into the cytoplasm, where, due to its nuclear localization
sequence, at least a fraction of it is immediately transported
back into the nucleus. Here, it can only be retained when
ssDNA (or ssRNA) is available as association site for the next
round of binding to single-stranded parts of newly formed
transcripts, and transient unwinding of unfavorable doublestranded regions of newly synthesized pre-mRNAs.
This model is in agreement with the finding that NDH II
binds to retroviral type D genomic RNA and migrates together
with it to the cytoplasm (Tang et al., 1997). However, from our
studies it is not yet clear whether NDH II recognizes specific
RNA sequences or structures. The model would also explain
the close proximity between NDH II and the transcriptionally
active RNA polymerase, as recently exemplified by showing
that NDH II acts as a ‘bridging factor’ between RNA
polymerase II and the transcriptional coactivator CBP/p300
(Nakajima et al., 1997). Indeed, we have biochemical evidence
that NDH II copurifies together with RNA polymerase II,
which indicates complex formation between these two
enzymes. Moreover, our model might not only hold true for
transcription of class II genes, but might be extended to the
synthesis of ribosomal genes, at least in specific cells and under
specific conditions (unpublished observations).
We are grateful to S. Monajembashi and G. Gothe for their
assistance in preparing microscopic photographs and figures, to A.
Willitzer for synthesis of the oligonucleotides, and to H.-P. Nasheuer
for many suggestions and critically reading the manuscript. The
monoclonal antibody against hnRNP A1 (4B10) was generously
provided by G. Dreyfuss (Howard Hughes Medical Institute,
University of Pennsylvania School of Medicine, Philadelphia). This
work was supported by grant Gr895/12-1 of the Deutsche
Forschungsgemeinschaft.
REFERENCES
Bass, B. L., Hurst, S. R. and Singer, J. D. (1994). Binding properties of newly
identified Xenopus proteins containing dsRNA-binding motifs. Curr. Biol.
4, 301-314.
Bernhard, W. (1969). A new staining procedure for electron microscopical
cytology. J. Ultrastruct. Res. 27, 250-265.
Bycroft, M., Grünert, S., Murzin, A. G., Proctor, M. and Johnston, D. S.
(1995). NMR solution structure of a dsRNA binding domain from
Drosophila staufen protein reveals homology to the N-terminal domain of
ribosomal protein S5. EMBO J. 14, 3563-3571.
Caceres, J. F., Misteli, T., Screaton, G. R., Spector, D. L. and Krainer, A.
R. (1997). Role of the modular domains of SR proteins in subnuclear
localization and alternative splicing specificity. J. Cell Biol. 138, 225-238.
Dreyfuss, G., Matunis, M. J., Piñol-Roma, S. and Burd, C. G. (1993).
hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62, 289321.
Eckmann, C. R. and Jantsch, M. F. (1997). Xlrbpa, a double-stranded RNAbinding protein associated with ribosomes and heterogeneous nuclear RNPs.
J. Cell Biol. 138, 239-253.
Gee, S., Krauss, S. W., Miller, E., Aoyagi, K., Arenas, J. and Conboy, J.
G. (1997). Cloning of mDEAH9, a putative RNA helicase and mammalian
homologue of Saccharomyces cerevisiae splicing factor Prp43. Proc. Nat.
Acad. Sci. USA 94, 11803-11807.
Grosse, F., Nasheuer, H. P., Scholtissek, S. and Schomburg, U. (1986).
Lactate dehydrogenase and glyceraldehyde-phosphate dehydrogenase are
single-stranded DNA-binding proteins that affect the DNA polymerase αprimase complex from calf thymus. Eur. J. Biochem. 160, 459-467.
Kramer, A. (1996). The structure and function of proteins involved in
mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367-409.
Kuroda, M. I., Kernan, M. J., Kreber, R., Ganetzky, B. and Baker, B. S.
(1991). The maleless protein associates with the X chromosome to regulate
dosage compensation in Drosophila. Cell 66, 935-947.
Ladomery, M., Wade, E. and Sommerville, J. (1997). Xp54, the Xenopus
homologue of human RNA helicase p54, is an integral component of stored
mRNP particles in oocytes. Nucl. Acids Res. 25, 965-973.
Lamond, A. I. and Earnshaw, W. C. (1998). Structure and function in the
nucleus. Science 280, 547-553.
Lee, C.-G. and Hurwitz, J. (1992). A new RNA helicase isolated from HeLa
cells that catalytically translocates in the 3′ to 5′ direction. J. Biol. Chem.
267, 4398-4407.
Lee, C.-G., Chang, K. A., Kuroda, M. I. and Hurwitz, J. (1997). The
NTPase/helicase activities of Drosophila maleless, an essential factor in
dosage compensation. EMBO J. 16, 2671-2681.
Li, H. and Bingham, P. M. (1991). Arginine/serine-rich domains of the su(wa)
and tra RNA processing regulators target proteins to a subnuclear
compartment implicated in splicing. Cell 67, 335-342.
McMillan, N. A. J., Carpick, B. W., Hollis, B., Toone, W. M., ZamanianDaryoush, M. and Williams, B. R. G. (1995). Mutational analysis of the
double-stranded RNA (dsRNA) binding domain of the dsRNA-activated
protein kinase, PKR. J. Biol. Chem. 270, 2601-2606.
Misteli, T. and Spector, D. L. (1998). The cellular organization of gene
expression. Curr. Opin. Cell Biol. 10, 323-331.
Molnar, G. M., Crozat, A., Kraeft, S. K., Dou, Q. P., Chen, L. B. and
Pardee, A. B. (1997). Association of the mammalian helicase MAH with
the pre-mRNA splicing complex. Proc. Nat. Acad. Sci. USA 94, 7831-7836.
Nakajima, T., Uchida, C., Anderson, S. F., Lee, C. G., Hurwitz, J., Parvin,
J. D. and Montminy, M. (1997). RNA helicase A mediates association of
CBP with RNA polymerase II. Cell 90, 1107-1112.
Ono, Y., Ohno, M. and Shimura, Y. (1994). Identification of a putative RNA
helicase (HRH1), a human homolog of yeast Prp22. Mol. Cell. Biol. 14,
7611-7620.
Pederson, T. (1998). Thinking about a nuclear matrix. J. Mol. Biol. 277, 147159.
Piñol-Roma, S. and Dreyfuss, G. (1991). Transcription-dependent and
transcription-independent nuclear transport of hnRNP proteins. Science 253,
312-314.
Piñol-Roma, S. and Dreyfuss, G. (1992). Shuttling of pre-mRNA binding
proteins between nucleus and cytoplasm. Nature 355, 730-732.
Richter, L., Bone, J. R. and Kuroda, M. I. (1996). RNA-dependent
association of the Drosophila maleless protein with the male X chromosome.
Genes Cells 1, 325-336.
Spector, D. L., Fu, X. D. and Maniatis, T. (1991). Associations between
distinct pre-mRNA splicing components and the cell nucleus. EMBO J. 10,
3467-3481.
Sukegawa, J. and Blobel, G. (1995). A putative mammalian RNA helicase
with an arginine-serine-rich domain colocalizes with a splicing factor. J.
Biol. Chem. 270, 15702-15706.
Tang, H., Gaietta, G. M., Fischer, W. H., Ellisman, M. H. and Wong-Staal,
F. (1997). A cellular cofactor for the constitutive transport element of type
D retrovirus. Science 276, 1412-1415.
Zhang, S. and Grosse, F. (1991). Purification and characterization of two
DNA helicases from calf thymus. J. Biol. Chem. 266, 20483-20490.
Zhang, S. and Grosse, F. (1994). Nuclear DNA helicase II unwinds both DNA
and RNA. Biochemistry 33, 3906-3912.
Zhang, S., Maacke, H. and Grosse, F. (1995). Molecular cloning of the gene
encoding nuclear DNA helicase II. J. Biol. Chem. 270, 16422-16427.
Zhang, S. and Grosse, F. (1997). Domain structure of human nuclear DNA
helicase II (RNA helicase A). J. Biol. Chem. 272, 11487-11494.