From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
Received for publication, August 5, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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We have previously shown that the N-terminal
domain of hepatitis delta virus (NdAg) has an RNA chaperone activity
in vitro (Huang, Z. S., and Wu, H. N. (1998)
J. Biol. Chem. 273, 26455-26461). Here we investigate
further the basis of the stimulatory effect of NdAg on RNA structural
rearrangement: mainly the formation and breakage of base pairs. Duplex
dissociation, strand annealing, and exchange of complementary RNA
oligonucleotides; the hybridization of yeast U4 and U6 small nuclear
RNAs and of hammerhead ribozymes and cognate substrates; and the
cis-cleavage reaction of hepatitis delta ribozymes were used to
determine directly the role of NdAg in RNA-mediated processes. The
results showed that NdAg could accelerate the annealing of
complementary sequences in a selective fashion and promote strand
exchange for the formation of a more extended duplex. These activities
would prohibit NdAg from modifying the structure of a stable RNA, but
allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a
more extensively matched target in cognate substrate. These and other
results suggest that hepatitis delta antigen may have a biological role
as an RNA chaperone, modulating the folding of viral RNA for
replication and transcription.
Hepatitis delta virus
(HDV)1 is a satellite virus
of hepatitis B virus. The genome of HDV comprises single-stranded
circular RNA of ~1700 nucleotides, and HDV RNA replicates through a
symmetrical rolling circle mechanism (1). Hepatitis delta antigen is
the only protein coded by HDV that is critical for viral replication (2) and virion assembly (3), although the molecular mechanisms have not
yet been elucidated. HDV RNA, of both genomic and antigenomic senses,
contains a ribozyme domain that can adopt a pseudoknot-like structure
and undergo cis-cleavage in vitro (4-7). The cis-cleaving activity is essential for generating monomeric size RNA molecules during viral replication (8, 9). Hepatitis delta antigen may enhance,
although it is not required for, the processing of multimeric size HDV
RNA in vivo (10). Conceivably, hepatitis delta antigen by
itself, or together with unidentified cellular factors, acts as an RNA
chaperone that modulates the ribozyme activity of HDV RNA.
RNA chaperones are defined as proteins that aid in the folding of RNA
by preventing misfolding or by resolving misfolded structures (11). The
RNA chaperone activities in vitro of several proteins that
interact with RNA with broad specificity have been explored through
their effect on ribozyme reactions. These proteins, including several
Escherichia coli ribosomal proteins (12), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 protein (13), and
the nucleocapsid protein of human immunodeficiency virus (14, 15), can
overcome the general limitations of ribozyme reactions and facilitate
ribozyme catalysis. Proteins with RNA chaperone activity are thought to
lower the activation energy necessary for breaking and reforming of
base pairs, although the molecular mechanism underlying RNA chaperone
activity is not known.
In vitro, hepatitis delta antigen can modulate the
cis-cleaving activities of HDV genomic RNA fragments and facilitate a
trans-acting hammerhead ribozyme to find its target in RNAs of various
sequences and lengths (16). Hepatitis delta antigen exerts its effect on these RNA-mediated processes by modifying the conformation of RNA
molecules and by promoting strand annealing and strand dissociation.
These properties of hepatitis delta antigen parallel many cellular
proteins and viral proteins with RNA/nucleic acid chaperone activity
(12-15). The functional domain of hepatitis delta antigen appears to
locate at the N-terminal region that is rich in basic amino acids and
contains the cryptic RNA binding domain, coiled-coil domain, and
nuclear localization signal (16). The core of the functional domain
overlaps with the coiled-coil domain, whereas the RNA binding domain
that binds HDV RNA specifically is not required for the RNA chaperone
activity (16).
Here we investigated further the basis of the stimulatory effect of the
N-terminal domain of hepatitis delta antigen, named as NdAg, on RNA
structural rearrangement. The results confirm and extend our previous
model. The results showed that NdAg could promote the annealing of a
variety of complementary sequences and stabilize RNA duplexes. NdAg
preferentially stimulated the formation of a more extended duplex among
competing sequences by facilitating strand annealing and strand
exchange in a selective fashion. Moreover, NdAg was able to facilitate
a trans-acting hammerhead ribozyme to discriminate a completely matched
from a non-completely matched target in a substrate RNA, but may not alter the structure of a stable RNA molecule. The selective strand annealing and selective strand exchange activity of NdAg may be important for modulating the folding of viral RNA for replication and transcription.
Proteins--
The N-terminal region (residues 1-88) of
hepatitis delta antigen, named as NdAg, was produced in E. coli BL21 (DE3) cells and purified by phosphocellulose column
chromatography as described previously (16). Fractions containing NdAg
were snap-frozen in liquid nitrogen and stored at Nucleic Acids--
RNA oligos 11, 14, 17, and 22 and DNA oligos
A, B, and C were chemically synthesized. Short unlabeled nucleic acid
oligos were used directly without purification. DNA oligos A and B were gel-purified. The concentration of each nucleic acid fragment was
determined by its absorbance at 260 nm. Carrier-free 5' end-labeled RNA
oligo 22 and DNA oligo C were made using [
HH16 and HH163 RNAs were synthesized by T3 RNA polymerase (Promega)
with synthetic DNAs as templates. HJ12L RNA, Rz1.H2 RNA, KSS3 RNA,
yeast U4 and U6 snRNAs, and P9 RNA were run-off transcripts of the T7
RNA polymerase (Promega) with restriction enzyme-linearized plasmid DNA
as templates. These RNAs were internally labeled by incorporating
[ Strand Annealing/Hybrid Formation
Assay--
Complementary RNA oligos, complementary DNA oligos, or U4
and U6 snRNAs were heated separately at 95 °C for 2 min, cooled to
room temperature, and incubated at the reaction temperature for at
least 5 min before use. In general, 10-µl reaction mixtures contained
indicated amounts of nucleic acid fragments and NdAg or other protein
in 1× reaction buffer (40 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.02 mM EDTA, 2% glycerol). Reactions
were performed at different temperatures; timings initiated by the
addition of NdAg or other protein unless otherwise indicated. Reactions
were terminated by transfer to ice, and the addition of 2 µl of
ice-cold stop solution (50 mM EDTA (pH 8.0), 2.5% SDS,
25% glycerol, 0.01% xylene cyanol, and 0.01% bromphenol blue).
Different nucleic acid species were resolved by electrophoresis through
a native polyacrylamide gel in TBE buffer (89 mM Tris-HCl,
89 mM boric acid, and 1 mM EDTA) with or
without 0.1% SDS at 4 °C. Gels were dried and examined by autoradiography.
Strand Exchange Assay--
The pre-annealed RNA duplex was made
by mixing 0.5 µM 32P-labeled oligo 22 with 2 µM complement in TE buffer. The RNA solution was heated
at 95 °C for 2 min, cooled slowly (~1 h) to room temperature, and
stored at Melting Temperature Determination--
Tubes
containing the pre-annealed RNA duplex in 10 µl of reaction buffer
were transferred to a PCR machine and incubated for 3 min at each
indicated temperature. One tube was removed at 5 °C intervals
through the temperature range indicated, and 2 µl of ice-cold stop
solution was added immediately to terminate the reaction. All of the
reaction mixtures were kept on ice before analysis. Electrophoresis of
the RNA samples and the subsequent data analysis were performed as
described for the annealing assay.
Ribozyme Reactions--
Hepatitis delta ribozymes HJ12L and
Rz1.H2 were heated at 95 °C for 2 min, cooled to room temperature,
and incubated at 37 °C for 5 min before use. RNA was pre-incubated
with NdAg in 1× reaction buffer for 30 min at 37 °C, and the
cis-cleavage reaction of HJ12L or Rz1.H2 was initiated by the addition
of MgCl2 to a final concentration of 12 mM. The
reaction was performed at 37 °C for various periods of time. Further
cis-cleavage was inhibited by the addition of an equal volume of quench
solution containing 50 mM EDTA, 7 M urea,
0.005% xylene cyanol, and 0.005% bromphenol blue. RNAs in the
ribozyme reaction mixture were analyzed on a 10% polyacrylamide, 7 M urea gel.
Hammerhead ribozyme and the KSS3 substrate RNA were separately heated
at 95 °C for 2 min and then cooled to the reaction temperature for
at least 5 min. The two RNAs were mixed and pre-incubated with NdAg in
1× reaction buffer for 30 min at 30 °C, and the trans-cleavage reaction was initiated by the addition of MgCl2 to a final
concentration of 12 mM. The reaction was performed at
30 °C for 15 min, after which samples were treated with 1 mg/ml
proteinase K at 37 °C for 25 min. RNAs in the ribozyme reaction
mixture were analyzed on a 15% polyacrylamide, 7 M urea
gel. The radioactivity of each RNA fragment was determined by
quantification using a PhosphorImager (ImageQuant; Amersham
Biosciences). The relative amount of each RNA fragment was
determined by dividing the radioactivity of the RNA fragment with the
number of C residues in the RNA molecule; thus, the fraction of RNA
undergoing cleavage was calculated.
Strategy--
Previously we had shown that the N-terminal domain
of hepatitis delta antigen (NdAg) modulates the cis-cleaving activity
of HDV genomic RNA fragments and activates the trans-cleavage reaction between hammerhead ribozymes and cognate substrates in vitro
(16). Conceivably, NdAg exerts its effect on RNA-mediated processes by
acting as an RNA chaperone, promoting RNA re-folding by facilitating the breakage and reforming of base pairs. To further investigate the
basis of RNA chaperone activity, purified NdAg was assayed for the
ability to promote the annealing of complementary sequences in RNA
oligos and complicated RNA molecules in dilute solutions, and to
accelerate strand exchange between an RNA duplex and a competing sequence.
Strand Annealing Activity of NdAg--
To assay the stimulatory
effect of NdAg on strand annealing, complementary oligos of low
concentrations were mixed, and duplex formation was monitored. In the
case of RNA oligos 22 (0.025 nM) and 11 (0.125 nM) (Fig. 1A),
there was only negligible duplex formation in the absence of NdAg, but
the extent and the rate of duplex formation were significantly elevated
in the presence of 1 µM NdAg (Fig.
2A). Thus, NdAg promoted the
annealing of complementary RNA oligos.
It is known that Mg2+ can neutralize the negative charge of
phosphate groups and stabilize the structure of RNA molecules. To address the question of the effect of Mg2+ on the strand
annealing activity of NdAg, we performed the strand annealing reaction
in the presence of MgCl2 and/or NdAg. We found that
MgCl2 of 10-40 mM alone facilitated the
annealing of RNA oligos 22 and 11, although its stimulatory activity
was significantly lower than that of NdAg. Nevertheless,
MgCl2 at this concentration range reduced, although it did
not eliminate, the strand annealing activity of 1 µM NdAg
(Fig. 2A and data not shown).
Annealing assays were done on other complementary nucleic acid pairs of
11-56 nt; NdAg facilitated the annealing of all nucleic acid pairs
tested, including DNA-DNA, DNA-RNA, and RNA-RNA pairs (data not shown).
In the case of DNA oligos A (43 nt), B (56 nt), and C (18 nt) (oligos A
and C are complementary to different regions of oligo B; Fig.
1B), NdAg greatly promoted the formation of the trimolecular
duplex and both bimolecular duplexes at low DNA concentrations (~0.5
nM) (B/C and B/CA duplex data are shown in Fig.
2B).
Furthermore, NdAg accelerated the hybridization of more complicated
RNAs in addition to simple complementary nucleic acid pairs. 50-500
nM NdAg facilitated 0.5 nM U4 snRNA and 2.5 nM U6 snRNA to form U4/U6 hybrid that potentially contains
two intermolecular base pairing regions (Figs. 1C and
2C). All of the aforementioned simple complementary nucleic
acid pairs hybridize spontaneously; however, U4 snRNA and U6 snRNA did
not hybridize spontaneously at even up to 25 nM in the
absence of NdAg (data not shown). This suggests NdAg influences the
ability of complex RNAs to interact. Therefore, in addition to short
complementary oligos, NdAg also promotes the annealing of the
complementary sequences reside in longer or more complicated RNAs.
Strand Annealing/Hybrid Formation in the Presence of
Non-homologous Nucleic Acid or Other Proteins--
To test whether
non-homologous nucleotide sequences perturb the acceleration of strand
annealing or hybrid formation stimulated by NdAg, we analyzed the
hybridization of a constant amount of U4 snRNA (0.5 nM
161-nt RNA, 80.5 nM nucleotide) and U6 snRNA (2.5 nM 112-nt RNA, 280 nM nucleotide)) in the
presence of increasing amounts of a non-homologous P9 RNA (a 160-nt
run-off transcript of pET15b vector) at 100, 250, or 500 nM
NdAg. As shown in Fig. 3A, for
a 360 nM nucleotide concentration of U4 snRNA (0.5 nM RNA, 80.5 nM nucleotide) and U6 snRNA (2.5 nM RNA, 280 nM nucleotide), U4/U6 hybridization
was not inhibited until the molar ratio of total nucleotide (the sum of
two snRNAs and P9 RNA) to NdAg exceeded 5:1, and at higher molar ratios
(>5:1), U4/U6 hybrid did not form. The result suggests that there is a
finite amount of NdAg available to facilitate duplex hybridization with
a limiting ratio of one NdAg to five nucleotides. To confirm this
finding, we performed strand annealing reactions of DNA oligos A and B
at low concentrations (total nucleotide was ~5 nM) in the
presence of a relatively high concentration of NdAg (1 µM) and increasing concentrations of three kinds of
nucleic acid: E. coli cellular RNA,
The acceleration of strand annealing/hybrid formation associated with
NdAg is not specific for any particular type of nucleic acid. Hence,
NdAg interacts with nucleic acid with broad specificity. We tested
whether two other general nucleic acid-binding proteins share similar
strand annealing properties with NdAg. We found that E. coli
SSB (single-stranded nucleic acid-binding protein) and T4 phage gp32
protein at a concentration range of 1-10 µM failed to
promote U4/U6 hybrid formation or to stimulate DNA or RNA duplex
formation (data not shown). In addition, the stimulatory effect of 50 nM NdAg on U4/U6 hybrid formation was not perturbed in the
presence of a 20-fold excess of T4 phage gp32 protein, a 50-fold excess
of E. coli SSB, or a 200-fold excess of bovine serum albumin
(Fig. 4). Thus, NdAg can facilitate
strand annealing/hybrid formation in the presence of these two nucleic
acid-binding proteins. These results indicate that the stimulation of
strand annealing is not a general property of nucleic acid-binding
proteins.
NdAg Favors Extended Duplex Formation by Facilitating Strand
Annealing and Strand Exchange in a Selective Fashion--
We
next examined the stimulatory effect of NdAg on the annealing of RNA
oligos of different lengths to a common sequence. RNA oligos 11, 17, and 22 were used for this study (Fig. 1A). Both oligos 11 and 17 are complementary to oligo 22. Oligo 17 has the same sequence as
oligo 11 but also has a 2-nt extension at the 5' terminus and a 3-nt
extension at the 3' terminus. Hence, oligo 17 can form a 17-base pair
duplex with oligo 22, whereas oligo 11 can form an 11-base pair duplex
with oligo 22. In the presence of 1 µM NdAg, the kinetics
of annealing of oligos 22 and 17, and oligos 22 and 11 were quite
similar, and the duplex formation reaction of both pairs of oligos
reached an equilibrium within 10 min at 30, 37, and 42 °C (data not
shown). The melting temperatures of the 22/11 duplex and 22/17 duplex
were ~50 and ~65 °C, respectively, under annealing reaction
conditions (Fig. 5A). The
competitive strand annealing reaction was performed in the presence of
0.025 nM 32P-labeled oligo 22 and 0.5 nM oligos 11 and 17 at temperatures below the
Tm of each duplex. In the absence of NdAg, only
a small fraction of oligos formed duplexes at 30, 37, or 42 °C.
Moreover, the amount of 22/11 duplex was slightly higher than that of
22/17 duplex at each temperature, and both duplexes were stable at
42 °C (Fig. 5B). In the presence of 1 µM
NdAg, all of oligo 22 participated in duplex formation. However, oligo
22 annealed more favorably to oligo 17 at 30 and 37 °C, and
exclusively to oligo 17 at 42 °C (Fig. 5B). Moreover, the
relative amount of 22/17 and 22/11 duplexes at each incubation
temperature was not changed when the reaction time varied between 10 and 180 min (data not shown). Thus, NdAg promotes the formation of a
more extended duplex among competing sequences, with the activity
higher at 42 °C than 30 and 37 °C. Therefore, hypothetically NdAg
facilitates strand annealing or stimulates duplex dissociation in a
selective manner.
We next monitored the ability of NdAg to promote strand exchange
between an RNA duplex and a competing RNA oligo (Fig.
6). The pre-annealed
32P-labeled 22/11 duplex (0.25 nM) was mixed
with a 100-fold excess of oligo 17 (25 nM). In the absence
of NdAg, the pre-annealed duplex gradually dissociated over a 30-min
period, with the released 32P-labeled oligo 22 concurrently
forming a duplex with oligo 17. Consequently, the
32P-labeled 22/11 duplex was converted to the
32P-labeled 22/17 duplex (Fig. 6A). In the
presence of NdAg, the conversion of 22/11 duplex to 22/17 duplex
occurred at an elevated rate at least during the first 10 min of the
reaction (Fig. 6A). Thus, in this experiment, NdAg
facilitated strand exchange. However, a strikingly different result was
obtained when the assay was performed using two other combinations of
pre-annealed RNA duplex and a competing RNA oligo in the presence of
NdAg. The data in Fig. 6 (B and C) show that
strand exchange between a pre-annealed 22/14 duplex (0.25 nM) and oligo 11 (25 nM) as well as between a
pre-annealed 22/17 duplex (0.25 nM) and oligo 11 (25 nM) was not observed in the presence of 1 µM
NdAg; each pre-annealed duplex was stabilized by NdAg instead. In the
absence of NdAg, there was a build-up of 22/11 duplex after 10 min,
whereas the conversion of the pre-annealed duplex to 22/11 duplex was
not completed. Because 22/14 duplex and 22/17 duplex contain more base
pairs than 22/11 duplex, NdAg appears to promote strand exchange only when the formation of a more extended duplex occurs. These results suggest that NdAg promotes strand exchange in a selective fashion, and
excludes the only role of NdAg in the strand exchange process as being
the promotion of the dissociation of the pre-annealed duplex.
Furthermore, the strand exchange reaction was also performed in the
presence of MgCl2 with or without NdAg. We found that 1 µM NdAg speeded up the strand exchange reaction between
22/11 duplex and RNA oligo 17 in the presence of 0-5 mM
MgCl2 but failed to facilitate the conversion of 22/11
duplex to 22/17 duplex in the presence of 10 or 20 mM
MgCl2 (Fig. 6D).
NdAg May Not Alter the Structure of a Stable RNA--
The studies
with the synthetic nucleic acid oligos disclose that NdAg promotes
strand annealing and strand exchange in a selective fashion, with this
property allowing NdAg to stimulate the formation of the most stable
duplex among complementary sequences. This finding led us to speculate
that NdAg could assist a contiguous sequence in adopting a stable
structure, which may not be biologically active, and that NdAg would
not alter the structure of a stable RNA. Here we investigated these
speculations by studying the effect of NdAg on an RNA that contains the
autolytic domain of the HDV genome and a cis-acting hepatitis delta
ribozyme mutant that has a more stable structure than its wild type
counterpart. Because hepatitis delta ribozyme has to adopt a specific
catalytic structure, the alteration of autocatalytic activity upon NdAg
treatment reflects ribozyme structure change.
HJ12L contains the autolytic domain of the HDV genome, and Rz1.H2 is a
hepatitis delta ribozyme mutant that has had its helix 1 substituted,
the helix 2 extended from 5 to 8 base pairs, and the helix 4 replaced
by a stable hairpin loop (Fig.
7A). Rz1.H2 cis-cleaved
efficiently and rapidly in the absence of NdAg, whereas HJ12L was much
less active under the same conditions (Fig. 7B). The
extended helix 2 rather than the alterations in helixes 1 and 4 of
Rz1.H2 may account for the elevated cis-cleaving activity under native
conditions, because we showed previously that the elongation of helix 2 enhances the resistance to formamide and stabilized the catalytic core
of hepatitis delta ribozymes (17). Moreover, in the experiment shown in
Fig. 7B, we found that pre-mixing with NdAg prior to the
initiation of cis-cleavage did not alter the cis-cleavage reaction of
Rz1.H2, whereas the same treatment attenuated the cis-cleaving activity
of HJ12L. The results indicate that NdAg modified the structure of
HJ12L but not Rz1.H2 and that the interaction with NdAg does not alter
the catalysis of Rz1.H2. The finding supports the hypothesis that NdAg
does not modify the structure of a stable RNA.
Effect of NdAg on the Reaction of a Trans-acting Hammerhead
Ribozyme--
We then used the trans-cleavage reactions of a
hammerhead ribozyme HH16 to further study the RNA chaperone activity of
NdAg. The trans-cleavage reaction of a hammerhead ribozyme involves at
least two steps. First, the ribozyme anneals to its target in a
substrate RNA for the assembly of a hammerhead catalytic domain; then,
cleavage occurs at a specific location by the catalysis of divalent
cations (18). KSS3 is a 107-nt RNA containing three targets for the
hammerhead ribozyme HH16 (Fig.
8A). The first target has a C
to A substitution; hence, upon binding to HH16, the helix I of the
reconstituted hammerhead catalytic domain contains an AG mismatched
pair. The second and the third targets are completely matched to HH16;
each can form 16 base pairs with HH16. HH163 is a mutant of HH16 and
has a G to U substitution to compensate for the mutation of the first
ribozyme target of KSS3 (i.e. no mismatch; Fig.
8A). We showed previously that NdAg promotes the trans-cleavage reaction of HH16 and all its targets in KSS3 under ribozyme excess conditions, with NdAg appearing to exert its effect by
elevating the mutual accessibility of two RNAs (16). Here, we found
that >95% of KSS3 was cleaved by HH16 and HH163 when 1 nM
KSS3 and 10 nM of each ribozyme were pre-incubated with 1 µM NdAg prior to the initiation of trans-cleavage (Fig.
8B). Furthermore, the major products of the two ribozymes
were the same; they were the 49-, 23/22-, and 13-nt RNAs, with none of
them containing any ribozyme target (Fig. 8B; the 22- and
23-nt RNAs were not resolved on the gel). Therefore, with an excess
amount of ribozyme and the facilitation of NdAg, each ribozyme target
of KSS3 is available for ribozyme binding and cleavage. The mismatched
base pair in the helix I region of the hammerhead catalytic domain does
not prevent cleavage under these reaction conditions.
We then used the same system to analyze the ability of NdAg to aid HH16
and HH163 in discriminating different ribozyme targets in KSS3 under a
condition with the ribozyme target in excess of the ribozyme. We
performed the trans-cleavage reaction of 1 nM KSS3 and 1 nM HH16 or HH163 (each KSS3 contains three ribozyme targets; therefore, the molar ratio of ribozyme target to ribozyme is
3). We found that the pre-incubation of two RNAs with 0.2 or 1 µM NdAg promoted trans-cleavage significantly; the
overall extent of cleavage of KSS3 was elevated from less than 5% to
>40% for each ribozyme. However, three ribozyme targets in KSS3 were
not equally accessible to each ribozyme. In the case of HH16, the amount of the 72-nt RNA, which contains the first target, was dominant
over the amount of all other cleavage products, indicating most of the
cleavage events occurred at the second and third targets that are
completely matched to HH16. In the case of HH163, the major cleavage
products were the 58-nt RNA, which contains the second and third
targets, and the 49-nt RNA, indicating the cleavage process occurred
predominantly at the first target that is completely matched to HH163.
These results illustrate that, when ribozyme is limiting, NdAg
selectively promotes the annealing of ribozyme to its completely
matched target in the substrate RNA.
We showed previously that NdAg is able to modify the structure of
HDV RNA and non-HDV RNAs (16). This property is known as RNA chaperone
activity (11). In this study we set up model systems to further analyze
the RNA chaperone activity of NdAg. We found that NdAg can facilitate
rearrangements of RNAs into more stable structures by selectively
accelerating the annealing of complementary sequences, as well as by
selectively promoting strand exchange.
How Does NdAg Stimulate Strand Annealing?--
Many nucleic acid
chaperones identified so far, such as the nucleocapsid protein of human
immunodeficiency virus (NCp7) (19, 20), the prion protein (PrP) (21),
the ORF1 protein of mouse LINE-1 retrotransposon (22), heterogeneous
nuclear ribonucleoprotein A1 (23, 24), and the major messenger
ribonucleoprotein particle protein p50 (25), facilitate the annealing
of a variety of complementary sequences. These proteins and NdAg do not
have a common structural domain or sequence motif for this strand
annealing activity except that most of them are rich in basic residues.
Basic residues can participate in nonspecific, electrostatic
interaction with nucleic acids, so the binding of NdAg and other
nucleic acid chaperones may facilitate complementary strand annealing
by charge shielding. Nevertheless, the extent of strand annealing
stimulated by each of these proteins appears to be too great to be
explained solely by a reduction in electrostatic repulsion.
NdAg contains the coiled-coil domain of hepatitis delta antigen. A
synthetic peptide How Does NdAg Facilitate the Formation of a More Stable
Structure?--
In this study we found that NdAg promotes RNA to adopt
a more stable structure via several methods. First, NdAg catalyzes annealing differentially among competing sequences; second, NdAg facilitates strand exchange selectively between a duplex and a single-stranded nucleic acid; and third, NdAg stabilizes a duplex if a
competing nucleic acid, for the formation of a more stable duplex, is
absent. Moreover, NdAg can modify the structure of short
oligonucleotides and long RNAs either intermolecularly or intramolecularly (this study and Ref. 16). We suspect that NdAg catalyzes selective strand annealing and selective strand exchange by
allowing the single strand The Effect of Mg2+ on the RNA Chaperone Activity of
NdAg--
In this report we showed that, in the presence of What Is the Possible Role of Hepatitis Delta Antigen in
Vivo?--
The finding that NdAg possesses RNA chaperone activity
strongly emphasizes the importance of this activity in the life cycle of HDV. Hepatitis delta antigen stimulates viral genome replication and
HDV mRNA transcription (27-29). One end of the rodlike structure of the HDV genome, which has a highly ordered structure conserved between HDV isolates, contains the promoters to direct the synthesis of
viral antigenomic RNA and the 0.8-kb mRNA (30-33). Because
hepatitis delta antigen stimulates the rearrangement of RNAs into more
stable structures, this protein may allow the HDV genome to adopt an active promoter structure to recruit cellular factors for replication and transcription.
The cis-cleaving activity of HDV RNA is involved in the processing of
multimeric RNA during viral replication (8, 9). The ribozyme domain
requires a pseudoknot structure for catalysis, but because of the
highly self-complementary nature of HDV RNA, the catalytically active
structure is not expected to exist in multimeric size RNA. How is the
ribozyme conformation formed on multimeric size RNA molecules? Because
hepatitis delta antigen can modulate the ribozyme activity of HDV
subgenomic fragments in vitro (16) and can enhance ribozyme
activity in vivo (10), hepatitis delta antigen appears to be
a facilitator of the processing reaction, although the mechanism
remains unsolved. Moreover, in addition to modulating the activity of
HDV ribozyme during viral replication, hepatitis delta antigen may
stabilize the HDV genome by preventing circular monomers from adopting
thermodynamically unstable and catalytically active pseudoknot structure.
The core of the RNA chaperone domain of hepatitis delta antigen has
been located at aa 24-59 (16). Therefore, the arginine-rich sequence
(aa 2-27) and the arginine-rich motifs (aa 97-107 and aa 136-146) of
hepatitis delta antigen that bind specifically to HDV RNA in a rodlike
structure in vitro (34, 35) are not required for the RNA
chaperone activity. However, these "HDV RNA binding motifs" may
allow hepatitis delta antigen to interact preferentially with HDV RNA
in virus-infected cells.
Applications--
In this report, we have demonstrated
that NdAg improves the specificity of hammerhead trans-acting
ribozymes. The selective strand annealing activity and the selective
strand exchange activity of NdAg may have other practical applications.
The ability of NdAg to promote the formation of a more stable duplex
might be employed to elevate the accuracy of nucleic acid annealing for primer extension reactions, polymerase chain reactions, reverse transcription reactions, and all kinds of hybridization reactions, such
as in situ, Southern, and Northern hybridizations. Whether NdAg is useful in these applications remains to be explored.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. The
concentration of NdAg was determined by ninhydrin assay with lysine as
standard. NdAg was diluted to the desired concentrations with protein
dilution buffer (50 mM Hepes-NaOH (pH 7.9), 1 M
NaCl, 1 mM EDTA (pH 8.0), and 20% glycerol) and used as
10× stock. T4 phage gene 32 protein (T4gp32) and E. coli
single-stranded DNA-binding protein (SSB) were purchased from Amersham Biosciences.
-32P]ATP
(Amersham Biosciences) and T4 polynucleotide kinase (New England
Biolabs). These 5' end-labeled nucleic acid fragments were purified
from polyacrylamide, 7 M urea gels, and their
concentrations were calculated from the radioactivity of each fragment
and the specific activity of [
-32P]ATP.
-32P]CTP to in vitro transcription
reactions and then purified from polyacrylamide, 7 M urea
gels. The concentration of labeled RNA was calculated from the
radioactivity of RNA and the specific activity of CTP. The cellular RNA
of E. coli BL21 (DE3) cells was isolated by TRIzol reagent
(Invitrogen) following the instructions from the manufacturer.
X174 virion ssDNA and
X174 replication form dsDNA were purchased
from New England Biolabs. Nucleic acid concentrations were determined
according to their absorbance at 260 nm. All nucleic acids were
resuspended in TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) and stored at
20 °C.
20 °C until used. The 32P-labeled
pre-annealed RNA duplex was diluted in TE buffer and mixed with or
without a competing RNA oligo, in the presence or absence of NdAg in 10 µl of reaction buffer at room temperature. Timing was initiated upon
the addition of NdAg, and the reaction was stopped by the addition of a
0.2 reaction volume of ice-cold stop solution. All of the reaction
mixtures were kept on ice before analysis. Electrophoresis of the RNA
samples and the subsequent data analysis were performed as described
for the annealing assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The nucleic acid molecules of this study.
A, RNA oligos 11, 14, 17, and 22. Oligo 22 is complementary
to the other three oligos. B, DNA oligos A, B, and C. Oligos
A and C are complementary to different regions of oligo B. C, U4 snRNA and U6 snRNA can form an intermolecular
duplex.
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Fig. 2.
Effect of NdAg on strand annealing/hybrid
formation. A, the annealing of RNA oligos. 0.025 nM 32P-labeled oligo 22 and 0.125 nM oligo 11 were mixed with 0 or 10 mM
MgCl2 in the absence or presence of 1 µM NdAg
in 1× reaction buffer (40 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.02 mM EDTA, 2% glycerol) at room
temperature for various periods. Samples were analyzed on an 18%
polyacrylamide, 0.1% SDS gel at 4 °C. B, the annealing
of DNA oligos. The reaction contains 0.5 nM
32P-labeled oligo C alone, 0.5 nM
32P-labeled oligo C and 0.4 nM oligo B, or 0.5 nM 32P-labeled oligo C and 0.4 nM
oligos A and B. The annealing reaction was performed in the absence or
presence of 0.5 µM NdAg in 1× reaction buffer at
37 °C for 20 min. Samples were resolved on a 15% polyacrylamide,
0.1% SDS gel at 4 °C. C, the hybridization of internally
labeled U4 and U6 snRNAs. 0.5 nM U4 snRNA and 2.5 nM U6 snRNA were incubated without any protein or with 50, 200, and 500 nM NdAg in 10 µl of 1× reaction buffer at
37 °C for 5 s, 30 min, 60 min, and 120 min. Mock
represents the control experiment with the protein fraction obtained
from the BL21(DE3) cells containing the empty pET15b vector. Samples
were resolved on a 7% polyacrylamide, 0.1% SDS gel at 4 °C.
Asterisks in these and subsequent figures indicate
radioactive labeling.
X174 ssDNA and
X174 dsDNA. As shown in Fig. 3B, B/A duplex formation was not affected by the E. coli cellular RNA or
X174 ssDNA
until the total nucleotide concentration exceeded 5 µM,
whereas the presence of
X174 dsDNA did not fully inhibit the
annealing of oligos A and B at even 12 µM total
nucleotide. These results support the notion that the binding site size
of an NdAg monomer is ~5 nucleotides, and suggests that complementary
strand annealing can occur in the presence of a 100-1000-fold excess
of non-homologous sequence as long as the amount of NdAg is sufficient
to coat the non-homologous sequences and the complementary nucleic acid
pair.
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Fig. 3.
NdAg facilitates strand annealing in the
presence of other nucleic acids. A, fixed amounts of U4
snRNA (0.5 nM RNA, 80.5 nM nucleotide) and U6
snRNA (2.5 nM RNA, 280 nM nucleotide) were
mixed with increasing amounts of non-homologous P9 RNA in the presence
of 0.1, 0.25, or 0.5 µM NdAg in 1× reaction buffer at
37 °C for 20 min. The molar fraction of U4/U6 duplex as a function
of [non-homologous nucleotide]/[total nucleotide of U4 and U6
snRNAs] ratio was plotted. B, 0.1 nM DNA oligo
A and 0.02 nM DNA oligo B were mixed with an increasing
concentration of E. coli cellular RNA (RNA),
X174 virion ssDNA (ssDNA), or
X174 replication form
DNA (dsDNA) in the presence of 1 µM NdAg in
1× reaction buffer at 37 °C for 60 min. The molar fraction of A/B
duplex as a function of the concentration of non-homologous nucleotide
was plotted. Data were collected from three independent
experiments.
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Fig. 4.
NdAg facilitates strand annealing in the
presence of other proteins. 0.5 nM internally labeled
U4 snRNA, 2.5 nM internally labeled U6 snRNA, and 50 nM NdAg were mixed with increasing amounts of T4 gp32,
E. coli SSB, or bovine serum albumin in 1× reaction buffer
at 37 °C for 20 min. Samples were analyzed on a 7% polyacrylamide,
0.1% SDS gel at 4 °C. means no protein;
1x, 10x, 20x, 50x, and
200x stand for 50 nM, 0.5 µM, 1 µM, 2.5 µM, and 10 µM
protein, respectively.
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Fig. 5.
A, the double strand to single strand
transition reaction of 22/17 and 22/11 duplexes. The 10-µl reaction
mixture contained 0.25 nM pre-annealed
32P-labeled 22/17 duplex in 1× reaction buffer. Incubation
was carried out at 25 °C for 3 min, and the temperature increased
every 3 min by an increment of 5 °C to a final temperature of
75 °C for 3 min. After each step of incubation, one tube was
transferred to ice and 2 µl of stop solution was added immediately.
Samples were analyzed on an 18% polyacrylamide, 0.1% SDS gel at
4 °C. B, NdAg promotes the formation of a more extended
duplex. 0.025 nM 32P-labeled oligo 22 and two
competing complementary oligos 11 and 17 (0.5 nM amounts of
each) were incubated with or without 1 µM NdAg in 1×
reaction buffer at the indicated temperature for 30 min. Samples were
analyzed on an 18% polyacrylamide, 0.1% SDS gel at 4 °C.
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Fig. 6.
Effect of NdAg and magnesium ion on strand
exchange. A pre-annealed 32P-labeled RNA duplex (0.25 nM) and an RNA oligo (25 nM) were mixed in the
absence or presence of 1 µM NdAg in 1× reaction buffer
at 30 °C for various periods (22/11 duplex and oligo 17 for
A, 22/14 duplex and oligo 11 for B, and 22/17
duplex and oligo 11 for C). The RNA duplex and RNA oligo
were heated at 95 °C for 5 min and then cooled to 50 °C for 5 min
in the absence of NdAg as the control. This does not necessarily bring
the system to equilibrium. For panel D, 0.25 nM
pre-annealed 32P-labeled 22/11 duplex and 25 nM
RNA oligo 17 were mixed with 0-20 mM MgCl2 in
the absence or presence of 1 µM NdAg in 1× reaction
buffer at 37 °C for 5 min. All samples were analyzed on a 15%
polyacrylamide, 0.1% SDS gel at 4 °C.
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Fig. 7.
Effect of NdAg on the
cis-cleavage reaction of HJ12L and Rz1.H2. A, the sequence
and proposed secondary structure of HJ12L and Rz1.H2. HJ12L
contains nt 683-770 of HDV genomic sense RNA; lowercase
letters were the sequence derived from the cloning vector
during in vitro synthesis. Rz1.H2 is a hepatitis delta
ribozyme. x in each RNA represents the cleaving point, and
H1-H4 are the four double-stranded regions of the ribozyme
molecule. B, each RNA was incubated with or without 240 nM NdAg in 1× reaction buffer at 37 °C for 30 min,
after which the cis-cleavage reaction was initiated by the addition of
12 mM MgCl2. Full-length RNA
(1o) was resolved from the 3' cleavage product
(3'P) on a 10% polyacrylamide-urea gel. The 5' cleavage
product ran off the gel.
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Fig. 8.
Effect of NdAg on the trans-cleavage reaction
of hammerhead ribozymes. A, hammerhead ribozymes HH16 and
HH163, and KSS3 substrate RNA. The underlined sequences in
KSS3 are the target of hammerhead ribozymes; 1,
2, and 3 indicate the cleavage sites.
B, cleavage of 1 nM KSS3 by 1 or 10 nM hammerhead ribozyme in the absence of NdAg or in the
presence of 0.2 or 1 µM NdAg in 1× reaction buffer.
Samples were analyzed on a 15% polyacrylamide, 7 M urea
gel. The overall extent of cleavage of KSS3 was calculated and
presented as percentage cleaved at the bottom of the gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-60 corresponding to aa 12-60 of hepatitis delta antigen forms anti-parallel coiled-coil dimers with its surface
lined with basic side chains, and
12-60 dimer can associate with
other dimers to form doughnut-like octamers (26). We speculate that
NdAg has a structure similar to that of
12-60 peptide and NdAg
binds nucleic acids as coiled-coil dimers in a nonspecific manner
mainly through charge-charge interactions. After this, a dimer may
interact with other dimers to form multimers of NdAg, consequently,
bringing complementary nucleic acid strands together via
protein-protein interaction. Alternatively, NdAg may form multimers
with each multimer interacting with several nucleic acid molecules
simultaneously. Because NdAg remains associated with nucleic acids
after facilitating strand annealing, nucleic acid molecules must be
able to change positions rapidly in the nucleic acid-NdAg complex to
find their pairing partners. Therefore, NdAg could facilitate annealing
by increasing the local concentration of complementary strands.
double strand transition to reach an
equilibrium more rapidly than normal. However, the molecular mechanisms
underlying the formation of a more stable structure remain to be
elucidated, e.g. because all short duplexes for the strand
exchange analysis of this study are perfect duplexes with single-stranded overhangs, factors that determine the efficiency of the
strand exchange process, such as internal mismatches, the length/sequence of the initial duplex and the role of the
single-stranded overhangs, remain to be identified. In addition,
whether the strand exchange process requires the complete dissociation
of the initial duplex or whether partial melting of the initial duplex
is sufficient to initiate strand exchange remains to be studied.
10
mM Mg2+, the strand exchange activity of 1 µM NdAg is attenuated. We suspect that NdAg may not exert
its effect on RNA structural modification in the presence of
10
mM Mg2+. This speculation is supported by our
previous observations; the ability of NdAg to modulate the cis-cleaving
activity of HDV subgenomic fragments and to stimulate the catalytic
activity of hammerhead trans-acting ribozymes relies on the
pre-incubation of NdAg with the relevant RNA molecules in the absence
of Mg2+ prior to the initiation of cleavage by the addition
of Mg2+ (16)2;
and 1 µM NdAg in conditions of limiting hammerhead
trans-acting ribozyme, with a Mg2+ concentration of 12 mM, resulted in a very low rate of hammerhead ribozyme
turnover, i.e. the release of cleavage products from ribozyme for the binding of new substrate (16). Thus, Mg2+
attenuates the RNA chaperone activity of NdAg.
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ACKNOWLEDGEMENTS |
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We thank Drs. S. C. Cheng, M. Lai, and M. S. Lin for discussion and for comments on the manuscript. We thank Dr. K. Deen for English editing.
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FOOTNOTES |
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* This work was supported by the Academia Sinica (Republic of China) and by Grants NSC-89-2311-B-001-077 and NSC-90-2311-B-001-013 from the National Science Council (Republic of China).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 886-2-27883134;
Fax: 886-2-27826085; E-mail: hnwu@gate.sinica.edu.tw.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M207938200
2 Z.-S. Huang and H.-N. Wu, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: HDV, hepatitis delta virus; SSB, single-stranded DNA-binding protein; aa, amino acid(s); nt, nucleotide(s); oligo, oligonucleotide; snRNA, small nuclear RNA; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.
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