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(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) ( 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
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. ( 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 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
Volume 270,
Number 34,
Issue of August 25, pp. 20084-20089, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)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).
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
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).
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, 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
HO, 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
, 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).
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).
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.
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.
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.
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.
)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.,
for example, has been known to bind to internal sites in the viral RNA
(Blumenthal and Carmichael, 1979; Meyer, 1981).
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.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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