©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Yeast Viral 20 S RNA Is Associated with Its Cognate RNA-dependent RNA Polymerase (*)

(Received for publication, May 5, 1995; and in revised form, June 19, 1995)

María P. García-Cuéllar (1) Luis M. Esteban (1)(§) Tsutomu Fujimura (1) Nieves Rodríguez-Cousiño (1) Rosa Esteban (1)(¶)

From the Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Salamanca 37071, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases.


INTRODUCTION

Most RNA viruses encode RNA-dependent RNA polymerases to replicate their RNA genomes. Since the host cells contain many cellular RNAs, the polymerase or polymerase machinery must find the proper viral RNA among them. This task could be crucial, particularly for the polymerases of (+) strand RNA viruses in which RNA polymerization reactions take place in non-subviral environments.

20 S RNA of the yeast Saccharomyces cerevisiae was first described by Kadowaki and Halvorson (1971a, 1971b) as a single-stranded RNA species induced in the starvation conditions required for the sporulation of diploid cells. Garvik and Haber(1978), however, showed that 20 S RNA is not related with the sporulation process because it is also induced in haploid cells. Its copy number reaches up to 100,000 copies per cell in the induction conditions. 20 S RNA is not infectious, and the horizontal transmission takes place only by the cytoplasmic mixing that occurs during mating or by cell fusion. There are no DNA copies of 20 S RNA in the yeast genome (Matsumoto et al., 1990); however, all 20 S RNA-carrying strains harbor a linear double-stranded RNA (dsRNA) (^1)called W (2.5 kilobases). Both 20 S RNA and W have been cloned, sequenced, and analyzed (Matsumoto and Wickner, 1991; Rodriguez-Cousiño et al., 1991; Rodriguez-Cousiño and Esteban, 1992). These studies indicate that W is the double-stranded form of 20 S RNA. 20 S RNA and the (+) strand of W have coding capacity for a protein of 91 kDa with sequences conserved among RNA-dependent RNA polymerases from (+) strand and double-stranded RNA viruses (Kamer and Argos, 1984; Argos, 1988; Poch et al., 1989). These conserved sequences are particularly similar to those of the beta-subunits of the replicases of RNA coliphages such as Qbeta (Rodriguez-Cousiño et al., 1991).

Some yeast strains also carry another single-stranded RNA, called 23 S RNA (Esteban et al., 1992). 23 S RNA (and its double-stranded RNA counterpart, T dsRNA) have also coding capacity for a protein of 104 kDa with motifs conserved in RNA-dependent RNA polymerases. p91 and p104, in turn, share a high degree of conservation that extends beyond the RNA polymerase consensus motifs, indicating that they are evolutionarily related. The 23 S RNA copy number is also induced in the starvation conditions. Thus, 20 S RNA and 23 S RNA form a new viral RNA family in yeast (Esteban et al., 1993). However, neither 20 S RNA nor 23 S RNA is encapsidated into viral particles. The double-stranded counterparts of 23 S and 20 S RNAs, T and W dsRNAs, were first described by Wesolowski and Wickner(1984) as independent replicons.

Recently, we have shown that 23 S RNA is associated with p104 in vivo and that this protein has single-stranded binding affinity in Northwestern assays (Esteban et al., 1994). In this report, we extend a similar analysis to 20 S RNA and found that 20 S RNA and its putative RNA polymerase p91 form a ribonucleoprotein complex. This protein-RNA interaction has specificity since p91 binds only 20 S RNA but not 23 S RNA. These results suggest that 20 S RNA utilizes p91 for its replication.


MATERIALS AND METHODS

Strains and Media

Yeast strains used are listed in Table 1. Media were as previously described (Wickner, 1978). Escherichia coli MV1190 (Bio-Rad) was used for the propagation of plasmids and as the host for the M13 helper phage K07 (Stratagene) to obtain single-stranded DNA. Expression of proteins from a pT7-7 vector (Tabor and Richardson, 1985) was carried out using E. coli BL21(DH3) (Studier and Moffatt, 1986).



Genetic Methods

Standard methods of genetic analysis were done according to Mortimer and Hawthorne(1975). To transfer a cytoplasmic genome from one haploid strain to another, we used kar1-1 mutants that are defective in nuclear fusion (Conde and Fink, 1976). The procedure called cytoduction (cytoplasmic mixing) was the same as that described by Wesolowski and Wickner (1984).

W dsRNA and 20 S RNA Preparation

To analyze the presence of W dsRNA in cells, we used the rapid method for extraction described by Fried and Fink(1978). 20 S RNA was prepared from 20 S RNA-induced cells as described (Rodriguez-Cousiño et al., 1991). 20 S RNA induction was done by transferring early stationary phase cells grown in YPAD medium (1% yeast extract, 2% peptone, 0.04% adenine sulfate, and 2% glucose) to 1% potassium acetate, pH 7.0, and incubating them at 28 °C for 16 h (Wejksnora and Haber, 1978). The RNAs were analyzed on native agarose gels. All buffers and solutions used to manipulate 20 S RNA were prepared in diethylpyrocarbonate-treated H(2)O to avoid RNase contamination.

Other Nucleic Acid Manipulations

Plasmid preparations, restriction enzyme digestions, DNA ligations, and transformations were as described by Maniatis et al.(1982). P-Labeled single-stranded RNA transcripts were made in vitro using T7 or T3 RNA polymerase following the recommendations of the supplier (U. S. Biochemical Corp.). Before the reaction, plasmid DNA was digested with appropriate restriction enzymes to obtain discrete DNA template fragments for run-off synthesis by T7 and T3 RNA polymerases.

Northern Blot Hybridization

The RNAs present in different yeast strains were analyzed by Northern blot hybridization. Double-stranded or single-stranded RNAs were separated in native agarose gels (1.5%) and denatured in the gel as previously described (Fujimura et al., 1990). Hybridization was done with P-labeled single-stranded RNA transcripts made in vitro with T7 or T3 RNA polymerases as mentioned above (Fujimura et al., 1990).

Plasmids

Plasmids pW1 and pLM53T were used to obtain strand-specific single-stranded RNA probes for Northern blot hybridization and have been previously described (Rodriguez-Cousiño et al., 1991; Esteban et al., 1994). Plasmid pW1 contains 1368 bases of W cDNA sequence from base 1017 (numbered from the known W 5`-end) to base 2374. pLM53T contains almost the entire known T cDNA sequence, a 2862-bp fragment from bp 8 to bp 2869 (the known T sequence is 2871 bp). Plasmid pPAZ1 was used to express a protein of 40 kDa (p40) with truncated amino acid sequences of the W-encoded protein, p91. It contains W cDNA sequences from bp 166 to 825 (amino acids 55-274) and from bp 1518 to 1878 (amino acids 505-625), cloned into the EcoRI site of pLM62 (Esteban et al., 1994), a derivative of pT7-7 vector (Tabor and Richardson, 1985). The protein was expressed in E. coli BL21(DE3) cells, and the purified p40 was used as antigen to raise polyclonal antibodies against the W-encoded protein (Fig. 1).


Figure 1: Diagramatic representation of W cDNA and the putative RNA-dependent RNA polymerase (p91) encoded in the W (+) strand (20 S RNA). The truncated protein p40 was expressed in E. coli cells and used to raise antibodies against p91. Amino acids (aa) from p91 present in p40 are indicated. Regions boxed and shaded in p91 are the amino acid consensus motifs conserved in RNA-dependent RNA polymerases according to Poch et al.(1989).



Antisera

The purification of protein p40 overproduced in E. coli BL21(DE3) cells and the preparation of antisera against p40 were as previously described (Esteban et al., 1994). Specific antibodies against p40 were purified from the sera following the method of Beall and Mitchell(1986).

Expression of p91

Yeast cells grown in different conditions were suspended in 1 M sorbitol, 125 mM potassium phosphate, pH 7.6, 20 mM 2-mercaptoethanol, 0.5 mg/ml zymolyase and incubated for 30 min at 30 °C to prepare spheroplasts. The spheroplasts were collected by low-speed centrifugation and suspended in lysis buffer containing 0.2% SDS, 50 mM NaCl, 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride (Sigma). Aliquots of the samples were treated with 10% trichloroacetic acid at 70 °C for 5 min and centrifuged. The precipitates were assayed for protein (Lowry et al., 1951). The rest of the samples were mixed with an equal volume of 2 SDS loading buffer and boiled for 5 min. Samples (30 µg of protein) were electrophoresed through a 10% polyacrylamide gel. Proteins were electroblotted onto a nitrocellulose sheet, and p91 was detected by Western analysis with specific antiserum, using the ``protoblot Western blot AP'' (Promega).

Sucrose Density Gradient Sedimentation

20 S RNA-induced cells were used to prepare cell lysates as previously described (Rodriguez-Cousiño et al., 1991). The lysates were either applied directly to a 10-40% (w/v) linear sucrose gradient prepared in buffer A (50 mM Tris, pH 7.6, 0.1 M NaCl, 30 mM MgCl(2), 100 µg/ml heparin, and 0.5 mM phenylmethylsulfonyl fluoride) or treated with 10 µg/ml RNase A for 15 min at 37 °C prior to centrifugation. Sedimentation was done in a SW28 rotor (Beckman) for 15 h at 25,000 rpm at 4 °C. Fractions were collected from each gradient. The nucleic acids present in each fraction were extracted and analyzed by agarose gel electrophoresis followed by Northern blot hybridization using a W (+) strand-specific probe. Proteins in the fractions were precipitated with 20% trichloroacetic acid, washed with 0.2% trichloroacetic acid, and then separated on an SDS-acrylamide gel, blotted onto a nitrocellulose filter, and detected with p91-specific antiserum.

Immunoprecipitation

p91 in a sucrose gradient was immunoprecipitated with specific antibodies using protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). The method was as described (Esteban et al., 1994). Proteins in the immunoprecipitate were separated directly on an SDS-polyacrylamide gel followed by Western analysis. The RNAs in the pellet were suspended in 50 µl of H(2)O, extracted once with a volume of phenol:chloroform, ethanol precipitated, and vacuum dried. Then, the RNAs were dissolved in 6 µl of 10 mM NaPO(4), pH 7.0, 1 M glyoxal and heated for 1 h at 50 °C to denature the RNAs. The sample was directly applied onto a nylon membrane, air dried, heated at 80 °C for 2 h under vacuum, and hybridized with the appropriate probes as described (Fujimura et al., 1990).


RESULTS

p91 Is Expressed in Yeast Cells

It has been shown (Rodriguez-Cousiño et al., 1991; Matsumoto and Wickner, 1991) that the (+) strand of W dsRNA (and 20 S RNA) has coding capacity for a protein of 91 kDa (p91) that spans almost the entire length of the molecule. This protein has sequences diagnostic of RNA-dependent RNA polymerases. Parts of the W cDNA sequence were cloned in an E. coli expression vector, and p40 (a truncated p91) was expressed (Fig. 1). The purified protein was used to raise polyclonal antibodies. p40 contains portions of the N terminus and C terminus of p91 but not the consensus motifs conserved in RNA-dependent RNA polymerases (see the shadedregions in Fig. 1). When crude extracts from different yeast strains were analyzed in a Western blot, the anti-p40 antibodies cross-reacted with a protein of about 91 kDa that was present only in W-carrying strains (Fig. 2C, lanes2 and 3). This protein was absent in a W-0 strain (Fig. 2C, lane1), suggesting that the protein of 91 kDa is the W dsRNA (20 S RNA)-encoded protein and that p91 is expressed in yeast cells. Hereafter, we call this protein p91. That p91 is really encoded in W was confirmed by the concomitant transference of W dsRNA and the ability to produce p91 by cytoplasmic mixing (cytoduction). The donor strain 37-4C (W) has a chromosomal mutation kar1-1 and is defective in nuclear fusion. By crossing the donor with the recipient strain AN33 (W-0), we obtained the cytoductant PAZ82, which has W dsRNA with the nucleus of AN33 (Fig. 2, A and B, lanes2). The anti-p40 antibodies now detected p91 in the cell lysate from the cytoductant (Fig. 2C, lane2).


Figure 2: Expression of p91 in W-carrying strains. A, ethidium bromide-stained agarose gel of total nucleic acids prepared from W and W strains. Strains AN33 (W, lane1), 37-4C (W, lane3), and PAZ82, a strain obtained by the transference of the cytoplasm of strain 37-4C into strain AN33 (lane2), were grown at 37 °C for 48 h on YPAD medium. Total RNAs were extracted and separated on a native agarose gel. The position of W dsRNA is indicated. T dsRNA, which moves slightly slower than W dsRNA in the gel, is also seen in lanes2 and 3. B, Northern blot analysis of total RNA from W-carrying strains. A Northern blot of the same gel shown in A was hybridized with a W (+) strand-specific probe made in vitro from plasmid pW1 with T3 RNA polymerase. The conditions used to grow the cells favor the accumulation of W dsRNA. C, Western blot analysis of proteins prepared from the same strains mentioned in A. Cells were grown to log phase in YPAD medium at 28 °C, and cell lysates were prepared as described under ``Materials and Methods.'' Proteins in the lysates were separated in an SDS-polyacrylamide gel and transferred onto a nitrocellulose filter. The W-encoded protein was detected with anti-p40-specific antiserum.



Strains 37-4C and PAZ82 also harbor T dsRNA (23 S RNA) in addition to W (Fig. 2, lanes2 and 3). The anti-p40 antibodies, however, did not cross-react with p104 (the putative RNA polymerase encoded in T dsRNA) present in the same cell lysates. Similarly, the anti-p104 antibodies used in previous studies do not cross-react with p91 either (Esteban et al., 1994; see also Fig. 6). This lack of cross-reactivity was expected since the truncated proteins used to raise these antisera did not have the consensus motifs conserved in RNA-dependent RNA polymerases.


Figure 6: p91 and p104 form ribonucleoprotein complexes specifically with their cognate RNAs. An aliquot of fraction 13 of the sucrose gradient shown in Fig. 4A, which contained 20 S and 23 S RNAs was incubated with anti-p91, anti-p104, or their respective preimmune sera. The immunoprecipitates were isolated and divided in two parts. One part was analyzed by SDS-gel electrophoresis followed by Western blotting. p91 and p104 in the precipitates were detected with anti-p91 (A, leftpanel) and anti-p104 antisera (A, rightpanel), respectively. RNA was extracted from the rest of the immunoprecipitates and subjected to dot blot analysis. 20 S and 23 S RNAs in the blots were detected by hybridization using 20 S RNA-specific (B, upperpanels) and 23 S RNA-specific (B, lowerpanels) probes, respectively. In the leftpanels in A and B, the gradient fraction was treated with anti-p91 antiserum (lane2) or its preimmune serum (lane1). In the rightpanels in A and B, the gradient fraction was treated with anti-p104 antiserum (lane2) or its preimmune serum (lane1).




Figure 4: p91 cosediments with 20 S RNA in sucrose gradients. A yeast lysate from strain 37-4C prepared as described under ``Materials and Methods'' was divided in two parts before centrifugation. One of them was loaded directly on a 10-40% sucrose gradient (A and B) and the other was treated with 10 µg/ml RNase A for 15 min at 37 °C prior to centrifugation (C and D). Fractions were collected from both gradients. The protein in the gradients was separated on SDS-polyacrylamide gels, and p91 was detected by Western blotting (A and C) using anti-p91 antiserum. The RNA in the gradients was separated on agarose gels, and 20 S RNA was detected by Northern blot hybridization (B and D) using a 20 S RNA-specific probe. The arrows on top of fraction 13 (panelsA and B) indicate the peaks of p91 and 20 S RNA, respectively, in the untreated sample. The arrow on top of fraction 16 (panelC) indicates the peak of p91 in the absence of 20 S RNA.



The anti-p40 antibodies did not cross-react with proteins smaller than p91. This suggests the absence of posttranslational proteolytic processing of p91. Consistently, we did not find amino acid sequences characteristics of viral proteases in p91 (Bazan and Fletterick, 1988; Rodriguez-Cousiño et al., 1991).

Expression of p91 under Different Growth Conditions

The copy number of 20 S RNA and W dsRNA are greatly induced under starvation conditions (shifting the cells from rich media to 1% potassium acetate, a poor carbon source and without nitrogen sources) and heat shock (growth at 37 °C), respectively. We studied the expression of p91 in various growth conditions. Cells from strain 37-4C were grown at 28 °C in rich YPAD media, and logarithmically growing cells were collected. The anti-p40 antiserum detected p91 easily in a cell lysate from these cells (Fig. 3A, lane1). When the cells were brought to stationary phase by further incubation in the same medium for another 32 h, the antiserum could barely detect p91 (lane2). Then, those stationary cells were transferred to 1% potassium acetate and incubated for 16 h (20 S RNA induction conditions). The amount of p91 increased to a level similar to that in log-phase cells (lane3). This suggests de novo synthesis of p91 during 20 S RNA induction, since control cells, which had been kept in the same YPAD medium instead of being transferred to 1% potassium acetate, had maintained the same, barely visible amount of p91 (lane4). As shown in Fig. 3B, the amount of 20 S RNA during the induction conditions increases dramatically compared to the one in logarithmically growing cells (more than 100-fold increase according to our rough estimation from Northern blots). These results indicate that the induction of 20 S RNA in potassium acetate media is not due to overexpression of p91. The copy number of W dsRNA can be increased by growing cells at 37 °C (Wesolowski and Wickner, 1984) (also compare Fig. 2, A and B, with Fig. 3B). Interestingly, the amount of p91 in W dsRNA-induced cells was similar to the one in growing cells at 28 °C (Fig. 3A, lanes1 and 5). These results suggest that the quantity (and possibly the quality) of p91 is regulated by the growth conditions of the host cells and that the higher level of p91 observed (Fig. 3A, lanes3 and 5) is a prerequisite for the induction of 20 S RNA and/or W dsRNA.


Figure 3: Expression of p91 in various growth conditions. A, Western analysis of p91 using anti-p40 antiserum. Cells from strain 37-4C were grown in YPAD complete medium at 28 °C and collected at different times: 16 h (log phase, lane1), 48 h (stationary phase, lane2), or 64 h (late stationary, lane4). 20 S RNA induction was done by transferring the stationary phase cells (grown in YPAD for 48 h at 28 °C) to 1% potassium acetate and incubating them for another 16 h (lane3). W dsRNA induction was done by growing the cells in YPAD at 37 °C for 48 h (lane5). As control, strain AN33 (W-0) was grown in YPAD for 16 h at 28 °C (laneC). Cell lysates were prepared as described under ``Materials and Methods'' and analyzed in an SDS-polyacrylamide gel. p91 was detected by Western blotting with anti-p40 antiserum. The amount of protein analyzed in each lane was normalized to 30 µg. B, nucleic acids were extracted from log phase cells or 20 S RNA-induced cells (growth conditions were as described in A) of strain 37-4C and analyzed on an agarose gel. The leftpanel shows the ethidium bromide staining of the gel. The rightpanel shows an autoradiogram of the Northern blot of the same gel hybridized with a 20 S RNA-specific probe. The band migrating between 20 S and 26 S rRNA seen in the leftpanel is 23 S RNA.



Association of p91 with 20 S RNA

When 20 S RNA and W dsRNA in crude cell extracts are analyzed by sucrose gradient centrifugation, their mobilities in the gradients are apparently not affected by pretreatment with phenol (Wesolowski and Wickner, 1984; Widner et al., 1991). In small RNA viruses, coat proteins usually provide the major part of the molecular masses of the virions (Matthews, 1991). Therefore, these data indicate that 20 S RNA and W dsRNA are not encapsidated into viral particles. However, there still remains the possibility that the RNAs are associated with a small number of protein(s). We examined the sedimentation of p91 in sucrose gradients. Crude extracts were prepared from 20 S RNA-induced cells and loaded on a linear 10-40% (w/v) sucrose gradient. As shown in Fig. 4A, p91 peaked at fractions 12-13 under our standard centrifugation conditions. When the same gradient was analyzed by a Northern blot, we found that 20 S RNA also peaked at fractions 12-13 (Fig. 4B). This suggests the physical association between p91 and 20 S RNA. To confirm their association, we conducted two experimental approaches. First, the crude extract was treated with RNase A and then separated in a sucrose gradient. As shown in Fig. 4C, p91 now sedimented at a slower rate upon 20 S RNA digestion and peaked at fraction 16. In our centrifugation conditions, bovine serum albumin (66 kDa) and catalase (240 kDa) sedimented in the gradient at fractions 20-21 and fractions 17-18, respectively. By extrapolation from these data, our rough estimation of the molecular mass of the 20 S RNA-free p91 is about 450 kDa. Thus, it could be an oligomeric form of p91 or p91 complexed with host proteins.

The second approach was immunoprecipitation of 20 S RNA with anti-p40 antisera (Fig. 5). Aliquots of fraction 13 from the sucrose gradient shown in Fig. 4, A and B, were incubated with anti-p40 antisera from two rabbits or with their preimmune sera, and then the immunocomplexes were isolated with protein A-Sepharose. As shown in Fig. 5B, the anti-p40 antisera effectively precipitated 20 S RNA (lanes2 and 4), while their preimmune sera did not (lanes1 and 3). When the sample was pretreated with phenol to eliminate proteins, both anti-p40 antisera did not immunoprecipitate 20 S RNA (lanes5 and 6). From these results we concluded that p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA.


Figure 5: 20 S RNA is immunoprecipitated by p91-specific antisera along with p91. Small aliquots of fraction 13 from the gradient shown in Fig. 4, A and B, were treated with anti-p91 antisera from two different rabbits immunized with the same protein antigen (p40) or with their preimmune sera. The immunocomplexes were purified with protein A-Sepharose CL-4B. Part of each sample was analyzed by Western blotting (A). The rest was subjected to dot blot analysis followed by RNA-RNA hybridization using a 20 S RNA-specific probe made from plasmid pW1 by T3 RNA polymerase (B). Lanes1 and 3, preimmune sera from two rabbits. Lanes2 and 4, antisera from the same rabbits after immunization with p40. Lanes5 and 6, the sample was phenol-extracted to remove proteins and then treated with the same antisera as in lanes2 and 4, respectively.



Specificity of p91-20 S RNA Complex Formation

Previously, we demonstrated that 23 S RNA forms a ribonucleoprotein complex with its putative RNA polymerase p104 (Esteban et al., 1994). Since 20 S RNA and 23 S RNA are closely related, we wished to answer the question whether p91 specifically forms a complex with 20 S RNA. As shown in Fig. 2, the anti-p91 antiserum (raised against p40) did not cross-react with p104. Similarly the anti-p104 antisera did not cross-react with p91 (Esteban et al., 1994) (see also Fig. 6). Therefore, if p91 forms complexes not only with 20 S RNA but also with 23 S RNA, the anti-p91 antisera should immunoprecipitate 23 S RNA in addition to 20 S RNA. As shown in Fig. 6, however, the anti-p91 antiserum immunoprecipitates 20 S RNA but not 23 S RNA from a sucrose gradient fraction, which contained both 20 S and 23 S RNAs. Similarly, the anti-p104 antiserum immunoprecipitated 23 S RNA but not 20 S RNA (Fig. 6B). Thus, these results indicate that 20 S RNA and 23 S RNA form ribonucleoprotein complexes only with their cognate RNA polymerases p91 and p104, respectively.


DISCUSSION

In this paper, we report the initial characterization of p91, the putative RNA-dependent RNA polymerase encoded in the (+) strand of W dsRNA (20 S RNA). This protein is present only in W-carrying strains and is cotransmitted along with W by cytoduction, confirming that it is cytoplasmically encoded and that it is really expressed in yeast cells. p91 is not processed proteolytically after translation.

The amount of p91 is apparently regulated by the growth conditions of the host cells. Stationary cells contain barely detectable amounts of p91, whereas growing cells or 20 S RNA-induced cells contain a higher amount of p91. The higher level of p91 in the latter case appears to be attained by de novo synthesis of p91. Although the levels of p91 in the growing cells and in 20 S RNA-induced cells are similar, their 20 S RNA contents are quite different. The amount of 20 S RNA in induced cells increases more than 100-fold as compared to that of growing cells (Fig. 3B). Since cells scarcely grow in 1% potassium acetate, the large increase of 20 S RNA copy number under the induction conditions may be due to the continuous synthesis of 20 S RNA without dilution of 20 S RNA by cell divisions. Alternatively, this large increase of 20 S RNA may be caused by a more active p91 polymerase in induced cells than in growing cells. In this context, it should be mentioned that p91 has a potential phosphorylation site by c-AMP-dependent protein kinases (Matsumoto and Wickner, 1991). Another possibility for the activation is that some host protein(s) necessary for the polymerase machinery is in limited concentrations in growing cells but abundant in induced conditions. Since 20 S RNA-free p91 obtained from 20 S RNA-induced cells moves as a molecule with an apparent molecular mass of 450 kDa (Fig. 4C), this could be a manifestation of the participation of a host protein(s) in the p91-20 S RNA ribonucleoprotein complex formation. If it is the case, it will provide valuable information on the viral-host interactions.

p91 forms a non-covalent ribonucleoprotein complex with 20 S RNA as demonstrated by their comigration in sucrose gradients (Fig. 4) and by immunoprecipitation of 20 S RNA with anti-p91 antisera (Fig. 5). This is quite analogous to the complex formation between 23 S RNA and p104 we recently reported (Esteban et al., 1994). Thus, their similarity in the complex formation adds another line of evidence to support the idea that 20 S RNA and 23 S RNA form an RNA viral family in S. cerevisiae.

Many laboratory yeast strains carry 20 S RNA, but only a few have 23 S RNA. And all 23 S RNA-carrying strains so far examined also harbor 20 S RNA. (^2)This might suggest that 23 S RNA depends on 20 S RNA for its replication. As shown in Fig. 6, however, these RNAs form complexes only with their respective cognate RNA polymerases. Since both RNAs have no coding capacity for proteins other than their polymerases, these results rather suggest that 20 S RNA and 23 S RNA replicate independently.

20 S RNA and 23 S RNA are not encapsidated into viral particles. The formation of complexes with their RNA polymerases could protect the RNAs in the cytoplasm from nuclease attacks. Similarly, the RNA polymerases p91 and p104 could gain the benefit of protection from proteases by complex formation. p91 appears to be more susceptible of proteolytic cleavage than p104 once it is released from the ribonucleoprotein complexes, since we frequently observed p91 more degraded than p104 in sucrose gradients after RNase treatment (Fig. 4C) (Esteban et al., 1994). As mentioned, these RNA polymerases, after RNA digestion, moved in sucrose gradients much faster than expected from their molecular masses (our rough estimations of their masses from mobilities in the gradients are 450 kDa for p91 and 500 kDa for p104). It could be indicative of the existence of homo-oligomeric forms for these proteins. Alternatively, they could form complexes with a cellular protein(s). p91 and p104 share a high degree of amino acid conservation (Esteban et al., 1992). Particularly, there are three regions well conserved outside the consensus sequences for RNA-dependent RNA polymerases. Thus, the same cellular protein(s) might interact with one of those regions in both polymerases to participate in the complex formation.

The host cytoplasms are crowded not only with proteins but also with many cellular RNAs. The complex formation of p91 (or p104) with 20 S RNA (or 23 S RNA) would relieve the polymerases from the hard task of finding their templates for replication. A high local concentration of the template toward the polymerase in the complex will not only ensure the specificity but also increase the efficiency of the reaction. Such complex formation between a viral RNA polymerase and its RNA genome could be seen as a more general phenomenon, especially among (+) strand RNA viruses, since their RNA polymerase reactions usually occur in non-subviral environments. The replicase of the coliphage Qbeta, for example, has been known to bind to internal sites in the viral RNA (Blumenthal and Carmichael, 1979; Meyer, 1981).

Recently, it has been reported that the dsRNA element NB631 (2.7 kilobases) present in some strains of the chestnut blight fungus Cryphonectria parasitica is closely related to T and W dsRNAs (Polashock and Hillman, 1994). The NB631 RNA is not encapsidated into viral particles, and it encodes a putative RNA-dependent RNA polymerase particularly similar to T- and W-encoded polymerases. Interestingly, the NB631 RNA is localized in mitochondria (Polashock and Hillman, 1994). T and W (and their single-stranded forms) are apparently localized in the cytoplasm since they show a 4:0 segregation pattern in meiosis, and they are transferred at high frequency during cytoduction. However, it is unlikely that T and W are localized in mitochondria. First, T and W can be maintained stably in ^0 strains, which are defective in the mitochondrial translational apparatus (Wesolowski and Wickner, 1984).^2 Second, yeast mitochondria preferentially utilize the UGA codon over UGG for tryptophan (Dujon, 1981) (this is one of the reasons why the mitochondrial localization of the NB631 RNA is claimed), whereas T and W do not use the UGA codon for tryptophan but for the termination of the polymerase genes. Therefore, if these RNAs are evolutionarily related, their localization within different compartments in their respective hosts can be considered as a good example of adaptability of RNA viruses. Considering the similarity among these RNAs, it is interesting to know whether the NB631 RNA or its single-stranded RNA counterparts form ribonucleoprotein complexes with the putative polymerase similar to those of p91-20 S RNA or p104-23 S RNA described here.


FOOTNOTES

*
This work was supported by Grants PB90-0998 and ACP94-0055 from the Dirección General de Investigación Científica y Técnica (Spain). 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.

§
Present address: Laboratory of Cellular and Molecular Biology, National Institutes of Health, Bethesda, MD 20892.

To whom correspondence should be addressed: Tel.: 34-23-120673; Fax: 34-23-267970.

(^1)
The abbreviations used are: dsRNA, double-stranded RNA; bp, base pair(s).

(^2)
M. P. García-Cuéllar and R. Esteban, unpublished observations.


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