©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Unusual Structure of a Novel RNA Replicon in Rice (*)

(Received for publication, January 23, 1995; and in revised form, May 19, 1995)

Toshiyuki Fukuhara Hiromitsu Moriyama Takeshi Nitta

From the Laboratory of Molecular Cell Biology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Several linear double-stranded RNA (dsRNA)()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).

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.


MATERIALS AND METHODS

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.


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.



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.


RESULTS

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).

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.

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).


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).




DISCUSSION

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: dsRNA, double-stranded RNA; kb, kilobase; kbp, kilobase pair; nt, nucleotide(s); ORF, open reading frame; MOPS, 3-(N-morpholino)propanesulfonic acid.


REFERENCES

  1. Allison, R., Johnston, R. E., and Dougherty, W. G.(1986)Virology 154, 9-20
  2. Boccardo, G., Lisa, V., Luisoni, E., and Milne, R. G.(1987)Adv. Virus Res. 32, 171-214 [Medline] [Order article via Infotrieve]
  3. Brown, G. G., and Finnegan, P. M.(1989)Int. Rev. Cytol. 117, 1-56 [Medline] [Order article via Infotrieve]
  4. Dodds, J. A., Morris, T. J., and Jordan, R. L.(1984)Annu. Rev. Phytopathol. 22, 151-168 [CrossRef]
  5. Fukuhara, T., Moriyama, M., Pak, J. Y., Hyakutake, H., and Nitta, T.(1993) Plant Mol. Biol. 21, 1121-1130 [Medline] [Order article via Infotrieve]
  6. Ghabrial, S. A. (1994)Adv. Virus Res.43,303-388 [Medline] [Order article via Infotrieve]
  7. Gubler, U., and Hoffman, B. J.(1983)Gene (Amst.) 25,263-269 [Medline] [Order article via Infotrieve]
  8. Hillman, B. I., Fulbright, D. W., Nuss, D. L., and Van Alfen, N. K. (1994) in Sixth Report of the International Committee for the Taxonomy of Viruses (Murphy, F. A., ed) Springer-Verlag New York Inc., New York
  9. Ishihara, J., Pak, J.-Y., Fukuhara, T., and Nitta, T.(1992)Planta 187,475-482
  10. Joklik, W. K. (1983) The Reoviridae, Plenum Publishing Corp., New York
  11. Mackenzie, S. A., Pring, D. R., and Bassett, M. J.(1988)Theor. Appl. Genet. 76, 59-63
  12. Mindich, L.(1988) Adv. Virus Res.35,137-176 [Medline] [Order article via Infotrieve]
  13. Moriyama, H., Nitta, T., and Fukuhara, T.(1995)Mol. & Gen. Genet. 248,in press
  14. Nuss, D. L., and Dall, D. J.(1990)Adv. Virus Res. 38, 249-306 [Medline] [Order article via Infotrieve]
  15. Pfeiffer, P., Jung, J. L., Heitzler, J., and Keith, G.(1993)J. Gen. Virol. 74, 1167-1173 [Abstract]
  16. Schuster, D. M., Buchman, G. W., and Rashtchian, A.(1992)Focus (Idaho) 14,46-52
  17. Shapira, R., Choi, G. H., and Nuss, D. L.(1991)EMBO J. 10, 731-739 [Abstract]
  18. Valverde, R. A., Nameth, S., Abdallha, O., Al-Musa, O., Desjardins, P., and Dodds, J. A. (1990)Plant Sci. (Shannon) 67,195-201
  19. Wakarchuk, D. A., and Hamilton, R. I.(1985)Plant Mol. Biol. 5, 55-63
  20. Wakarchuk, D. A., and Hamilton, R. I.(1990)Plant Mol. Biol. 14, 637-639 [Medline] [Order article via Infotrieve]
  21. Wang, A. L., and Wang, C. C.(1991)Annu. Rev. Microbiol. 45, 251-263 [CrossRef][Medline] [Order article via Infotrieve]
  22. Zabalgogeazcoa, I. A., and Gildow, F. E.(1992)Plant Sci. (Shannon)83,187-194

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.