(Received for publication, May 5, 1995; and in revised form, June 19, 1995)
From the
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.
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) ()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
-subunits of
the replicases of RNA coliphages such as Q
(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.
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).
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).
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.
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.
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. ()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 Q,
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 strains, which are defective
in the mitochondrial translational apparatus (Wesolowski and Wickner,
1984).
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.