(Received for publication, January 23, 1995; and in revised form, May 19, 1995)
From the
A linear, plasmid-like, double-stranded RNA (dsRNA) was isolated
from rice, and its entire sequence of 13,952 nucleotides (nt) was
determined. The dsRNA encodes a single, unusually long, open reading
frame (13,716 nt, 4,572 amino acid residues), which includes an RNA
helicase-like domain and an RNA-dependent RNA polymerase-like domain. A
series of Northern hybridization and primer extension experiments
revealed that the coding (sense) strand of the dsRNA contains a
discontinuity (nick) at a position 1,211 nt (or 1, 256 nt) from the 5`
end. This discontinuity divides not only the coding strand of dsRNA
molecule into a 1,211-nt fragment and a 12,741-nt fragment (or a 1,
256-nt fragment and a 12, 696-nt fragment) but also divides the long
open reading frame into a 5` part of 1,045 nt (348 amino acid residues)
and a 3` part of 12,671 nt (4,224 amino acid residues) or a 5` part of
1,090 nt (363 amino acid residues) and a 3` part of 12,626 nt (4,209
amino acid residues). It seems likely that almost all dsRNA molecules
in rice plants contain such a discontinuity. This rice dsRNA appears to
be a novel and unique RNA replicon. Several linear double-stranded RNA (dsRNA) We found a large dsRNA in both cultivated rice (Oryza sativa L.) and wild rice (Oryza rufipogon, an ancestor of O.
sativa). This rice dsRNA has the following plasmid-like properties
(Fukuhara et al., 1993), which differ from those of
conventional plant RNA viruses: 1) plants harboring the dsRNA are
symptomless, 2) its inheritance is vertical, 3) it is present at a
constant concentration, and 4) it does not seem to be associated with
distinct virus-like particles. The entire sequence of 13,952 nt of
the rice dsRNA was determined from a series of independent and
overlapping cDNA clones. To our knowledge, ours was the first report of
the entire nucleotide sequence of a plasmid-like RNA replicon from a
plant. Furthermore, although many kinds of plant RNA virus have an RNA
genome, there are few reports of large (more than 10 kbp) plant RNA
viruses. The coding (sense) strand of the rice dsRNA molecule contains
a single, long ORF of 13,716 nt (4,572 amino acid residues), and the
ORF appears to be the longest found to date in plant genes. No
nucleotide and amino acid sequences with extensive homology to those of
this dsRNA and of this ORF were found in a search of data bases, but an
RNA helicase-like domain and an RNA-dependent RNA polymerase-like
(replicase-like) domain were detected within the ORF (see Fig. 1; Moriyama et al. (1995)). In addition to the
four above mentioned interesting properties, the most conspicuous
molecular feature of rice dsRNA is its single, unusually long ORF.
Indeed, the rice dsRNA can be regarded as a novel RNA replicon in
plants.
Figure 1:
Genetic organization of the rice dsRNA
and a map showing the cDNA clones that were used as probes for Northern
hybridization. Hel and Pol indicate an RNA
helicase-like domain and an RNA-dependent RNA polymerase-like domain,
respectively. The nucleotide sequence data will appear in the GSDB,
DDBJ, EMBL, and NCBI nucleotide sequence data bases with accession
number D32136.
We now report another unusual structural feature of this
dsRNA molecule; the coding (sense) strand of the dsRNA molecule
contains a discontinuity (nick) at a specific position. The biological
implication of the discontinuity is discussed.
Figure 2:
Detection of a 1.3-kb RNA. A,
results of electrophoresis on a nondenaturing agarose gel of dsRNA
samples that were prepared from 10 individual rice plants. Similar
amounts of dsRNA were obtained from the five individual rice plants. B and C, results of Northern hybridization of rice
dsRNAs. The samples were the same as in A but were subjected
to electrophoresis on a denaturing agarose gel. The (±) DNA
probes derived from clones F76 (B) and F88 (C) were
used. A band of about 1.3-kb was detected in each of the five samples
when F76 was used as probe.
Figure 3:
Characterization of a 1.3-kb RNA by a
series of Northern hybridization experiments. A, results of
Northern hybridization of rice dsRNAs fractionated on a nondenaturing
agarose gel with the (±) DNA derived from each cDNA clone (R201,
F76, F88, or R306) as probe. B, results of Northern
hybridization of rice dsRNAs, fractionated on a denaturing agarose gel.
Each of four kinds (R201, F76, F88, and R306) of the (±) DNA
probe, the(-) riboprobe, and the (+) riboprobe was
used.
When the dsRNA
fractionated on a denaturing agarose gel, the hybridization profile
obtained with each probe was different (Fig. 3B). When
R201, which corresponds to the 5`-terminal region of the long ORF (Fig. 1), was used, the 14- and 1.3-kb bands together, the 14-kb
band alone, and the 1.3-kb band alone were detected with the (±)
probe, the(-) probe, and the (+) probe, respectively. When
F76 was used, the 14-kb band superimposed on the 12.7-kb band (a
composite band) plus the 1.3-kb band, the 14-kb band alone, and the
12.7- and 1.3-kb bands together were detected with the (±)
probe, the(-) probe, and the (+) probe, respectively. It was
confirmed by electrophoresis under different conditions from those used
to generate Fig. 3B that the composite band consisted
of the 14- and 12.7-kb bands (data not shown). When we used either F88
or R306, which corresponds to the 3`-terminal region of the long ORF (Fig. 1), similar hybridization profiles were obtained. The
composite band consisting of 14- and 12.7-kb RNAs, the 14-kb band alone
and the 12.7-kb band alone were detected with the (±) probe,
the(-) probe, and the (+) probe, respectively. These
results indicate that the coding strand of the dsRNA molecule contains
one discontinuity (nick) at a position about 1,300 nt from the 5` end
and that the dsRNA molecule is separated into three molecules, a 1.3-kb
molecule (the 5` part of the coding strand), a 12.7-kb molecule (the 3`
part of the coding strand), and a 14-kb molecule (the entire noncoding
strand) on denaturing agarose gels. Furthermore, almost all the dsRNA
molecules found in almost all rice plants are likely to contain such a
discontinuity, because 1) only the 1.3-kb band (not the 14-kb band) was
detected using the (+) probe derived from R201 (Fig. 3B), 2) the dsRNA samples analyzed in Fig. 3B were isolated from seedlings that were
comprised of many rice plants, and 3) the intensity of the
hybridization signal of the 1.3-kb band in Fig. 2B was
similar for each of the five samples.
Figure 4:
The
exact position of the discontinuity within the coding strand of the
rice dsRNA molecule. Results of a primer extension experiment designed
to determine the exact position of the discontinuity within the coding
strand of the rice dsRNA molecule. Lanes G, A, T, and C show sequencing ladders as size markers, and lane P shows the products of the primer extension reaction (arrowheads).
At least two possible mechanisms can be proposed for the
formation of this discontinuity; the coding strand of the dsRNA could
be cleaved either by its own ribozyme activity (self-cleavage) or by a
specific ribonuclease encoded by the host genome. Pfeiffer et
al. (1993) recently reported that a 16.7-kbp dsRNA from Vicia
faba contains one discontinuity, which seems to divide the coding
strand into molecules of 12.2 and 4.5 kb. The biological implications
of the discontinuity in dsRNA from rice and V. faba are
unknown, but the finding of a discontinuity in different kinds of dsRNA
molecules suggests that such a discontinuity plays an important role in
the life cycle of these large plasmid-like dsRNAs. The discontinuity is
likely to affect the replication of the dsRNA molecule, the
transcription of the coding strand, and the translation of the long
ORF. Although other large dsRNAs with plasmid-like properties similar
to those of the rice dsRNA have been found in barley (Zabalgogeazcoa
and Gildow, 1992), pepper (Valverde et al., 1990), Phaseolus vulgaris (Wakarchuk and Hamilton, 1985; Mackenzie et al., 1988), and so on, it is not yet known whether these
large dsRNAs include a discontinuity. The rice dsRNA has some
plasmid-like properties and encodes one extremely long ORF that
contains an RNA helicase-like domain and an RNA-dependent RNA
polymerase-like domain (Fukuhara et al., 1993; Moriyama et
al., 1995). Comparisons of the amino acid sequences of these two
domains and of the entire genetic organization of the rice dsRNA to
those of potyviruses (single-stranded RNA viruses with a (+) RNA
genome of about 10 kb (Allison et al., 1986)) and the 12.7-kbp
dsRNA of chestnut blight fungus, Cryphonectria parasitica, (Cryphonectria hypovirus 1-713 (Hillman et al.,
1994), previously known as hypovirulence-associated virus (Shapira et al., 1991)) suggest that the rice dsRNA is evolutionarily
located between potyviruses and Cryphonectria hypovirus
1-713 (Moriyama et al., 1995). Furthermore, the rice
dsRNA contains a discontinuity at a specific position in the coding
strand. Many kinds of dsRNA virus have been detected in bacteria
(Mindich, 1988), protozoa (Wang and Wang, 1991), fungi (Ghabrial,
1994), plants (Nuss and Dall, 1990), and animals (Joklik, 1983), but no
dsRNA virus that contains a discontinuity within its genome, except for
the V. faba dsRNA, has been reported. Thus, the rice dsRNA
appears to be a unique and novel RNA replicon.
(
)have been detected in various apparently healthy
plants, from algae (Ishihara et al., 1992) to higher plants
(Dodds et al., 1984; Brown and Finnegan, 1989). These dsRNAs
are generally classified into two groups (Brown and Finnegan, 1989).
One group consists of low molecular weight dsRNAs (1.0-5.0 kbp)
that are associated with proteins to form virus-like particles, and
some of which are referred to as cryptoviruses (Boccardo et
al., 1987). The second group consists of high molecular weight
dsRNAs (more than 10 kbp) that do not appear to be associated with
virus-like particles. Because no designation for these high molecular
weight dsRNAs has yet been agreed upon, these dsRNAs have been
variously referred to as RNA plasmids (Brown and Finnegan, 1989),
indigenous dsRNAs (Valverde et al., 1990), and enigmatic
dsRNAs (Wakarchuk and Hamilton, 1990; Fukuhara et al., 1993).
Isolation of dsRNA
Double-stranded RNA was
purified from young leaves (or seedlings) of rice plants (Japonica rice
cv. Nipponbare) as described previously (Fukuhara et al.,
1993).Probes for Hybridization
The cDNA clones (F76 and
F88, Fig. 1) and the reverse transcriptase polymerase chain
reaction clones (R201 and R306, Fig. 1) were obtained by the
method of Gubler and Hoffman(1983) and the 5` rapid amplification of
cDNA ends method (Schuster et al., 1992), respectively. These
clones were used as probes for Northern hybridization. (±) DNA
probes were made with a random primer DNA labeling kit (Takara, Kyoto,
Japan) and [-
P]dCTP (Amersham Corp.) and
used for Northern hybridization as shown in Fig. 2and Fig. 3. cDNA fragments were inserted into pBluescript II
SK(+) (Stratagene, La Jolla, CA), and then recombinant plasmids
were linearized with a restriction enzyme (BamHI or EcoRI). (+) and(-) riboprobes were made by in
vitro transcription in a system that contained T3 or T7 RNA
polymerase (Boehringer Mannheim) and
[
-
P]UTP (Amersham Corp.). (+)
and(-) riboprobes were used in Fig. 3B.
Northern Hybridization
The dsRNAs were subjected
to electrophoresis either on 1.0% native agarose gels that contained 40
mM Tris acetate buffer (pH 8.1), 2 mM EDTA, and 500
ng/ml ethidium bromide (see Fig. 2A and 3A) or
on 1.2% denaturing agarose gels that contained 20 mM MOPS
buffer (pH 7.0), 5 mM sodium acetate, 1 mM EDTA, 0.66 M formaldehyde, and 500 ng/ml ethidium bromide (see Fig. 2, B and C, and 3B). The dsRNAs
on native or denaturing gels were transferred to nylon membranes
(Zeta-Probe GT membrane; Bio-Rad). Hybridization was performed in
hybridization medium for the DNA probes (250 mM sodium
phosphate (pH 7.2), 1 mM EDTA, 7% SDS, 1% bovine serum
albumin, and 1% Nonidet P-40) for 16 h at 65 °C or in the medium
for the riboprobes (125 mM sodium phosphate (pH 7.2), 250
mM NaCl, 7% SDS, and 50% formamide) for 16 h at 60 °C. The
membranes were washed twice with 20 mM sodium phosphate buffer
(pH 7.2) containing 5% SDS for 30 min and washed twice again with the
same buffer containing 1% SDS for 30 min at 65 °C.Primer Extension
The 25-mer oligonucleotide
(5`-GCAATAGCATCGGATCTTAAATGTG-3`) that was complementary to positions
1,461- 1,485 from the 5` end of the coding strand of the dsRNA
molecule was synthesized as a primer for primer extension analysis. The
extension reaction was carried out in 50 mM Tris-HCl (pH 8.3),
75 mM KCl, 3 mM MgCl, 10 mM dithiothreitol, 2.5 µM dATP, dGTP, and dTTP, 1
pmol/µl primer, 0.1 µg/µl heat-denatured rice dsRNA as
template, 10 units/µl SuperScript II reverse transcriptase (Life
Technologies, Inc.), and 0.037 MBq/µl
[
-
P]dCTP (Amersham Corp.) for 2 min at 25
°C. 1 µl each of 10 mM dATP, dCTP, dGTP, and dTTP were
added to 9 µl of the reaction mixture, and then the reaction was
continued for 1 h at 37 °C. The reaction products were subjected to
electrophoresis on a 5% polyacrylamide gel that contained 89 mM Tris, 89 mM borate, 2 mM EDTA, and 50% urea.
Detection of a 1.3-kb RNA
When we tried to
isolate dsRNA from 10 separate rice plants (Japonica rice cv.
Nipponbare) that grew in a greenhouse, dsRNA at similar levels was
detected in the case of five individuals by native agarose gel
electrophoresis and staining with ethidium bromide (Fig. 2A). Five of the ten plants did not contain the
dsRNA. There was no obvious difference between the two groups of
plants. These samples of dsRNA were subjected to electrophoresis on a
denaturing agarose gel, and then hybridization experiments were
performed using two cDNA probes (F76 and F88; the locations of these
probes within the rice dsRNA are indicated in Fig. 1). Bands of
14-kb RNA were found, as expected, in the five samples with either F76
or F88 (Fig. 2, B and C), and, in addition, a
band of RNA of about 1.3 kb was unexpectedly detected with F76 only,
and the intensity of the hybridization signal of the 1.3-kb band was
similar for each of the five samples (Fig. 2B). These
bands of 1.3-kb RNA were not detected by Northern hybridization with
F88 as probe (Fig. 2C) or by electrophoresis on
nondenaturing agarose gels and staining with ethidium bromide (Fig. 2A).Characterization of 1.3-kb RNA
In order to
characterize the 1.3-kb RNA in further detail, we performed additional
Northern hybridization experiments. As well as F76 and F88, clones R201
and R306 were used as a source of probes (Fig. 1). Three types
of P-labeled probe were prepared: a coding (sense)
strand-specific riboprobe ((+) probe), a noncoding (antisense)
strand-specific riboprobe ((-) probe), and a DNA probe made from
both strands as template ((±) probe). Furthermore, the dsRNAs
were subjected to electrophoresis on either native or denaturing
agarose gels. Whenever the dsRNA was fractionated on a native agarose
gel, only the band of 14-kbp RNA was detected with each type of
(±) probe (Fig. 3A).
The Exact Position of the Discontinuity (Nick)
To
determine precisely the site of the discontinuity on the coding strand
of the dsRNA molecule, we synthesized a 25-mer oligonucleotide primer
that was complementary to positions 1,461-1,485 nt, counted from
the 5` end of the coding strand of the dsRNA molecule, and we performed
a primer extension experiment. Two main products of the primer
extension reaction were detected in lane P in Fig. 4.
This indicates that the discontinuity was located between A at position
1,211 and C at position 1,212 and/or between A at position 1,256 and G
at position 1,257 from the 5` end of the coding strand. This
discontinuity divides not only the coding strand of the dsRNA molecule
into a 1,211-nt 5` fragment and a 12,741-nt 3` fragment (or a 1,256-nt
5` fragment and a 12,696-nt 3` fragment) but also divides the long ORF
into a 5` part of 1,045 nt (348 amino acid residues) and a 3` part of
12,671 nt (4,224 amino acid residues) (or a 5` part of 1,090 nt (363
amino acid residues) and a 3` part of 12,626 nt (4,209 amino acid
residues)). We tried to confirm the 5` end of 12.7-kb molecule of the
coding strand by the 5` rapid amplification of cDNA ends method
(Schuster et al., 1992), and the results were similar to those
of the primer extension experiments (data not shown).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.