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Molecular and Cellular Biology, November 1999, p. 7461-7472, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Point Mutations in Yeast CBF5 Can
Abolish In Vivo Pseudouridylation of rRNA
Yeganeh
Zebarjadian,1
Tom
King,2
Maurille J.
Fournier,2
Louise
Clarke,1 and
John
Carbon1,*
Department of Molecular, Cellular and
Developmental Biology, University of California, Santa Barbara,
California 93106,1 and Department of
Biochemistry and Molecular Biology, University of Massachusetts,
Amherst, Massachusetts 010032
Received 25 February 1999/Returned for modification 15 April
1999/Accepted 29 July 1999
 |
ABSTRACT |
In budding yeast (Saccharomyces cerevisiae), the
majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to
direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is
the most likely pseudouridine (
) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known
synthase, and shares the "KP" and "XLD" conserved sequence
motifs found in the catalytic domains of three distinct families of
known and putative
synthases. To gain additional evidence on the
role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative
synthase domain of Cbf5p. Yeast strains expressing these mutated
cbf5 genes in a cbf5
null background are
viable at 25°C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of
in rRNA
at extreme temperatures. Substitution of alanine for an aspartic acid
residue in the conserved XLD motif of Cbf5p (mutant
cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some
of the mutants are temperature sensitive both for growth and for
formation of
in the rRNA. In most cases, the impaired growth
phenotypes are not relieved by transcription of the rRNA from a
polymerase II-driven promoter, indicating the absence of polymerase
I-related transcriptional defects. There is little or no abnormal
accumulation of pre-rRNAs in these mutants, although preferential
inhibition of 18S rRNA synthesis is seen in mutant
cbf5D95A, which lacks
in rRNA. A subset of mutations in
the
synthase domain impairs association of the altered Cbf5p
proteins with selected box H/ACA snoRNAs, suggesting that the
functional catalytic domain is essential for that interaction. Our
results provide additional evidence that Cbf5p is the
synthase
component of box H/ACA snoRNPs and suggest that the pseudouridylation
of rRNA, although not absolutely required for cell survival, is
essential for the formation of fully functional ribosomes.
 |
INTRODUCTION |
In eukaryotes the biosynthesis of
rRNA occurs in a specialized organelle known as the nucleolus (33,
41, 46, 56). rRNA is transcribed by RNA polymerase I (Pol I) as a
single large precursor, which undergoes a series of endo- and
exonucleolytic cleavages to produce mature rRNA species. In the yeast
Saccharomyces cerevisiae, the 35S pre-rRNA precursor is
processed to produce mature 18S, 5.8S, and 25S RNAs (54).
The 5S rRNA and ribosomal proteins are imported into the nucleolus for
assembly into precursors of the 40S and 60S ribosomal subunits before
their export to the cytoplasm (16, 41, 46). An interesting
feature of rRNA maturation is the extensive modification the 35S
precursor undergoes prior to subsequent cleavage events (29, 40,
39). One such modification, isomerization of uridine to
pseudouridine (
), is by far the most abundant posttranscriptional
modification of rRNA (29, 40, 39). Formation of
is also
known to occur in tRNAs (49), small nuclear RNAs (snRNAs),
and small nucleolar RNAs (snoRNAs) (17, 30). Despite the
abundance of
in various classes of RNA, very little is known about
its role in RNA structure and function.
The conversion of uridine to pseudouridine is thought to involve the
breakage of the N1 glycosidic bond, the rotation of the base 180°
around the N3-C6 axis, followed by reformation of a covalent bond at
position C5 (15, 39). The isomerization reaction is
catalyzed by a group of enzymes known as
synthases. A limited number of
synthases have been identified in both prokaryotes and
yeast and have been shown to have enzymatic activity in vivo (reviewed
in reference 40). Comparative sequence analysis has further identified a number of putative
synthases that share the KP
and XLD (where X stands for T, A, or R residues) sequence motifs found
in three distinct families of known and putative
synthases
(24). Recently, it has been demonstrated that the aspartic
acid in the XLD motif is absolutely essential for the catalytic
activity of truAp, an Escherichia coli tRNA synthase that
catalyzes the conversion of uridine to
at positions 38, 39, and 40 in tRNA (20). Mutation of this residue inhibits
formation in tRNA in vitro.
Prokaryotic
synthases have a high degree of site-specificity in
vivo, both for tRNA and rRNA substrates (40, 43). However, site selection for pseudouridylation in eukaryotic rRNAs involves a
class of small nucleolar RNAs (snoRNAs) known as box H/ACA snoRNAs (2, 12, 13, 40, 48, 52). The majority of characterized yeast
H/ACA snoRNAs have been shown to act as guides to direct site-specific
pseudouridylation of rRNA. These snoRNAs form two short regions of
complementary to rRNA, resulting in an unpaired pocket that is thought
to render a specific uracil residue accessible to the rRNA
synthase. All known guide H/ACA snoRNAs achieve this
complementarity through a common hairpin-hinge-hairpin-tail secondary structure motif. The consensus folding includes two evolutionary conserved elements, box H (defined as AGA in yeast and 5'-ANANNA-3' in other species) located in the hinge region, and box ACA, positioned exactly 3 nucleotides (nt) from the 3' end of
all H/ACA snoRNAs (2). That box H/ACA elements play important roles in site selection was demonstrated by mutational analysis. The site of modification is almost exclusively 14 to 16 nt
from the H or ACA box (12, 13, 36). These conserved elements
also play a role in snoRNA synthesis and are believed to serve as
binding sites for specific proteins (2, 31).
Two yeast H/ACA snoRNAs, snR30 and snR10, are important for normal
processing of pre-rRNA (35, 50, 51). Shutdown of snR30
biosynthesis results in defective synthesis of mature 18S rRNA and is
lethal (3, 35). However, there is no evidence for
involvement of this snoRNA in
formation. Strains lacking snR10 are
defective in the processing of the 35S pre-rRNA precursor and exhibit a
cold-sensitive phenotype (50). Additionally, snR10 is known
to direct synthesis of
in the core of the peptidyltransferase center (36). Interestingly, the remaining 18 H/ACA guide
RNAs examined are individually dispensable, although each specifies
formation at one or two specific sites in rRNA (36, 52, 44).
The H/ACA snoRNAs are found as RNP particles (snoRNPs) in the
nucleolus. The most recent characterization of the H/ACA snoRNP complexes by two studies has provided some insight into the functions of the protein cofactors (19, 58). These studies have shown independently that all H/ACA snoRNAs form a stable complex with the
essential nucleolar proteins Gar1p, Nhp2p, Nop10p, and Cbf5p. Gar1p,
the best-characterized member of this complex, is required for stable
association of the box H/ACA snoRNAs with the pre-rRNA (2, 12,
13) and is necessary for pre-RNA processing (13, 14).
Mutations in Gar1p also inhibit
formation in rRNA (8). Moreover, there is evidence from coimmunoprecipitation experiments (25) for interaction of Gar1p with Cbf5p. Nhp2p was
initially characterized as an HMG-like protein (23) and was
subsequently shown to be related to the known RNA binding protein,
ribosomal protein L32 (55, 19). Nhp2p stably associates with
all H/ACA snoRNAs (19, 58) in accordance with its putative
RNA binding activities. Nop10p is also required for the stability of
the H/ACA snoRNPs, and depletion of Nhp2p or Nop10p results in
defective 18S pre-rRNA processing and disruption of
formation in
rRNA (19).
The fourth protein constituent of the H/ACA snoRNPs, Cbf5p, has been
postulated to have several in vivo functions. Cbf5p was initially
isolated as a low-affinity centromere DNA binding protein in vitro
(22). CBF5 interacts genetically with the
centromere-binding protein gene CBF2/NDC10 and with the
meiosis-specific protein kinase gene MCK1 (21).
In addition, Cbf5p binds microtubules in vitro, which is consistent
with a role in centromere function (22). Interestingly,
Cbf5p also functions in ribosome biogenesis. We have shown previously
that the temperature-sensitive mutation cbf5-1 prevents rRNA
transcription at the nonpermissive temperature and reduces the
cytoplasmic pool of 40S and 60S ribosomal subunits (9). In
addition, overexpression of SYC1/RRN3, an RNA Pol I-specific transcription factor (59), suppresses the cbf5-1
conditional growth defects (9). Cbf5p is also implicated in
rRNA processing because depletion of this protein causes defects in 18S
rRNA maturation (25). Moreover, there is evidence that Cbf5p
could be an rRNA
synthase. Cbf5p and its protein homologs rat NAP57
(32), Drosophila Nop60B (42), and
human dyskerin (18) have sequence homology to E. coli truBp (24), which catalyzes the conversion of
uridine to
at position 55 in tRNA (38). Furthermore,
Cbf5p shares the KP and XLD motifs found in three distinct families of
known and putative
synthases (24). Like Gar1p, Nhp2p,
and Nop10p, Cbf5p coimmunoprecipitates with all members of the H/ACA
class of snoRNAs, and its depletion inhibits
formation in rRNA
(25). However, to date, there is no direct evidence that any
of these proteins, including Cbf5p, functions as rRNA
synthases.
In this study, we demonstrate that alteration of selected highly
conserved amino acids in the putative
synthase domain of Cbf5p
abolishes in vivo pseudouridylation of rRNA. This loss of
correlates with a slow-growth phenotype and abnormally reduced levels
of cytoplasmic 40S and 60S subunits. Our results indicate that
Cbf5p-dependent pseudouridylation is essential for normal growth of
yeast cells; strains lacking
in rRNA are viable but display severe
growth defects.
 |
MATERIALS AND METHODS |
Plasmids and yeast strains.
Plasmids used are as follows:
pBluescript II KS(
) (Stratagene, San Diego, Calif.); pBFG
(10) (2µm-derived plasmid with LEU2 marker, and
three hemagglutinin [HA] epitope tags expressed under control of the
PGK1 promoter); pYHY18-CBF5 (21);
pCBF5-BFG (the entire CBF5 open reading frame
[ORF] was PCR amplified and cloned into the EcoRI and
XhoI sites of pBFG; this plasmid complements the
cbf5-null allele); and pNOY103 (2µm-derived plasmid,
URA3 marker, with the 35S rRNA gene expressed from the
GAL7 promoter [37]), a gift from M. Nomura,
University of California, Irvine. Plasmids pcbf5D65A-BFG,
pcbf5P67A-BFG, pcbf5L94A-BFG and
pcbf5D95A-BFG are described below.
The following yeast strains were used: YPH274
(MATa/MAT
ade2-101/ade2-101
his3-200/his3-200 leu2-1/leu2-1 lys2-801/lys2-801 trp1-1/trp1-1
ura3-52/ura3-52 [Yeast Genetic Stock Center]); YWJ64-ts (same
genotype as YPH274 plus cbf5-1/cbf5-1) (9);
YCC131 (MATa ade2-1 can1-100 his3-11 leu2-3,112
trp1-1 ura3-1
cbf5::TRP1/pYHY18-CBF5); YCC133,
which is isogenic with YCC131 except that it carries
pCBF5-BFG; YCC37 (MAT
ade2-101ochre
his3-
200 leu2-
1 lys2-801amber trp1-
1 ura3-52
cbf5::HIS3/p64-FAT10) (42); YCC35,
which is isogenic with YCC37 except that it carries pFLY64-ADNS (2µm
plasmid, LEU2 marker, with Nop60B cDNA fused to the
ADH1 promoter) (42); YHY64
1/pNOY103
(MAT
ade2-101 cbf5-1 his3-
200 leu2-1 lys2-801 trp1-1
ura3-52/pNOY103) (strain YHY64
1 [21] was
transformed with plasmid pNOY103 [37]); and YZCC12-2
(ade2-101 his3-
200 leu2-3,112 syc2::HIS3
trp1-
901 ura3-521/pBFG). The mutant strains cbf5D65A, cbf5P67A, cbf5L94A, and
cbf5D95A were constructed for this study (see below).
Mutagenesis.
The mutations D65A, P67A, L94A, and D95A were
generated by oligonucleotide-directed in vitro mutagenesis by using the
Sculptor Mutagenesis Kit (Amersham, Arlington Heights, Ill.). The
CBF5 ORF was released from pCBF5-BFG as an
EcoRI/XhoI fragment and subcloned into
pBluescript II KS(
), previously linearized with the same restriction
enzymes and blunt ended with the Klenow DNA Pol I and deoxynucleoside
triphosphates (dNTPs). Single-stranded phagemid DNA (1 µg/µl) was
annealed to 1.6 pmol of each of the following mutagenic primers per
µl: D65A (TGGAAGGTTTAGCTAGATTAATGAC), P67A (GATGGGTTGGAAGCTTTATCTAGA),
L94A (TTTTGGATAGCTGTACCAGAGTG-3'), and D95A
(GTAACTTTTGGAGCCAATGTACCAG-3').
Primers were synthesized by Genosys Biotechnologies (Woodlands,
Tex.). The sequence of each mutagenic primer is complementary to the
CBF5 gene, except for the targeted bases (denoted by
boldface lettering). Mutations either introduced a restriction site
(underline) or destroyed a natural site (italic type). A
HindIII restriction site was introduced by a single base
change in P67A; a PvuII site was introduced by a triple base
change in L94A, while XbaI and BamHI sites were destroyed in D65A and D95A, respectively. Mutations are denoted by the
original amino acid and position in the Cbf5p peptide sequence, followed by the substituted amino acid, which in all four cases is
alanine. Each mutated sequence was checked by restriction digestion and
DNA sequencing before it was subcloned into the pBFG vector. Mutated
plasmids were introduced into the yeast strain YCC131 by using the
Alkali Yeast Transformation Kit (Bio 101, Inc., La Jolla, Calif.), and
transformants were plated onto a medium lacking leucine. Strains that
have lost the wild-type YHY18-CBF5 plasmid were selected on
5-fluoroorotic acid plates containing uracil as described previously
(47). Plasmids were reisolated from the transformants and
digested with appropriate restriction enzymes to check the integrity of
each construct.
Northern blot hybridizations.
Total yeast RNA was isolated
as described previously (27). Approximately equal aliquots
of RNA preparations were separated in 1.2% agarose-formaldehyde gels,
transferred to nylon membranes for 24 h by capillary action, and
probed with individual 32P-labeled oligonucleotides
complementary to various regions of the 35S pre-rRNA transcript as
described previously (26).
3H pulse-chase labeling of rRNA.
Pulse-chase
labeling of pre-rRNA was done essentially as described previously
(9, 53), except that cultures were maintained at 30°C or
shifted to 37°C for 2 or 10 h prior to labeling. RNA samples
isolated from aliquots containing approximately equal numbers of cells
were separated on 1.2% agarose-formaldehyde gels, transferred to nylon
membranes, treated with En3Hance as described by the
supplier (DuPont/NEN), and exposed to X-ray film. The band intensities
were quantitated with the Alpha Innotech Digital Imager, version 3.1 (Alpha Innotech Corp., San Leandro, Calif.).
Immunoprecipitations and Western blots.
Whole-cell protein
extracts were prepared by glass bead lysis in a
radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl
sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride).
Immunoprecipitations, SDS-polyacrylamide gel electrophoresis, and
immunoblotting were carried out by using an immunoprecipitation kit
(Promega, Madison, Wis.) according to the manufacturer's protocols.
Monoclonal antibody HA.11 (Covance-Babco, Richmond, Calif.) at 5 µg/ml was used for immunoprecipitation of HA-tagged wild-type and
mutant proteins. However, a 1:80 dilution of the antibody was used for
immunodetection of the HA epitope on Western blots. After
immunoprecipitation of H+ACA snoRNPs, the sedimented beads were
resuspended in 300 µl of immunoprecipitation lysis buffer and
deproteinized with 1.5 mg of proteinase K (Boehringer Mannheim,
Indianapolis, Ind.) per ml for 30 min at 37°C. Subsequently, total
RNA from each sample was prepared as described earlier (2)
and analyzed on 8% acrylamide-7 M urea gels (45). RNA was
transferred to a nylon membrane by electrophoresis (11).
Oligodeoxyribonucleotides (18 to 20 bp) complementary to yeast snR8,
snR10, snR31, and snR37 were used as hybridization probes. A 50-pmol
mixture of the oligonucleotides was end labeled with polynucleotide
kinase (Promega) and 150 µCi of [
-32P]ATP (Amersham)
as described previously (45).
Analysis of
in rRNA.
Control (YCC133) and experimental
yeast strains harboring various cbf5 mutant alleles were
pregrown at 30°C in yeast extract-peptone containing 2% glucose
(YPD) to an optical density at 660 nm (OD660) of 1.0. The
cells were pelleted, washed with sterile water, and resuspended in
fresh YPD at an OD660 of 0.2. After incubation with shaking
at either 30 or 37°C for 60 min (1.5 h shift) or 7 h (10 h
shift), cells were pelleted, washed with sterile water, resuspended in
25 ml of low-phosphate medium (57) at an OD660 of 0.5, and incubated for an additional 30 min (1.5 h shift) or 3 h (10 h shift) at either 30 or 37°C with agitation. The culture was
centrifuged, and the cells were resuspended and incubated at the
permissive or nonpermissive temperature in 3 ml of the same medium
containing 1 mCi of [32P]orthophosphate (900 mCi/mmol)
for 60 min (1.5-h shift) or 90 min (10-h shift). Total RNA was
extracted and fractionated by electrophoresis in 1.0%
agarose-formaldehyde gels. The 18S and 25S rRNAs were isolated from the
gel by electroelution, ethanol precipitated, and digested with RNase
T2 (Sigma R3751) in 5 µl of 50 mM ammonium acetate (pH
4.5)-0.05% SDS-1 mM EDTA for 90 min at 37°C. Aliquots of digested
RNA corresponding to about 17,000 or 100,000 cpm, depending upon the
experiment, were subjected to two-dimensional thin-layer chromatography
(TLC) on plastic-backed cellulose plates (EM Science no. 5577) by using
isobutyric acid-NH4OH-H2O (577:38:385, by
volume) in the first dimension and 2-propanol-HCl-H2O (70:15:15, by volume) in the second dimension. Patterns were analyzed with a Molecular Dynamics PhosphorImager, and Up:
p ratios were determined with ImageQuant v1.1 software. Results are derived from a
total of three to four TLC plates per strain, corresponding to at least
two independent experiments.
Sucrose gradient sedimentation profiles of cytoplasmic ribosomal
subunits.
The protocol was essentially as described previously
(1, 9), except that the cultures were examined 2 h
after being shifted to the nonpermissive temperature (37°C). The cell
extracts were fractionated by sedimentation through 7 to 47% linear
sucrose gradients prepared in 50 mM Tris acetate (pH 7.0)-0.5 M
KCl-12 mM MgCl2-1 mM dithiothreitol, conditions which
cause complete dissociation of ribosomes into the 40S and 60S subunits.
 |
RESULTS |
Substitution of conserved amino acid residues in the putative
synthase sequence domains impairs in vivo function of yeast Cbf5p.
Cbf5p has sequence homology to known and putative
synthases
(24) and is known to occur in H/ACA snoRNP complexes
(25, 58). Thus, Cbf5p is likely to be the enzyme responsible
for catalyzing
formation in rRNA. Previous studies demonstrated that depletion of Cbf5p results in loss of pseudouridylation of rRNA in
vivo (25). However, depletion of several other protein components of the H/ACA snoRNPs also results in the loss of
in rRNA
(8, 19, 58). If Cbf5p is the enzyme responsible for
conversion of U to
in rRNA, then single amino acid substitutions in
the highly conserved
synthase sequence motifs would be expected to
greatly reduce or eliminate
in rRNA.
Both the KP and XLD motifs (where X stands for R, A, or T) are
conserved among three families of known and putative

synthases
(
24) and are thought to play important roles in catalysis
(Fig.
1A). D65 (immediately adjacent to
the KP motif) is more conserved
among the truB family of synthases, to
which Cbf5p belongs. The
aspartic acid in the XLD motif is the only
residue conserved in
the fourth family, represented by the
E. coli truA synthase. Recently,
mutation of this particular amino
acid residue was shown to abolish
the

synthase activity of truAp
(
20). To address the role of
Cbf5p in rRNA
pseudouridylation, alanine substitutions were introduced
by
site-directed mutagenesis at positions D65 (immediately adjacent
to the
KP motif), P67 (within the KP motif), and L94 and D95 (within
the
"XLD" motif) (Fig.
1B).

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FIG. 1.
Regions of yeast Cbf5p subjected to site-directed
mutagenesis. (A) A schematic of the overall sequence of yeast Cbf5p is
shown, indicating the location of the truB homology region (shaded box)
and the KKE/KKD repeat motif (unshaded box) (22). The highly
conserved synthase domains in Cbf5p and representatives of the four
known synthase families (24) are shown. Domains I and II
correspond to motifs I and II described by Koonin (24). The
highly conserved KP and XLD sequence motifs in domains I and II are
indicated in boldface type. E. coli truA contains no
apparent homology to domain I. Numbers in parentheses indicate the
number of amino acids between the domains or, in the case of truA, to
the N terminus. (B) The putative synthase catalytic domains in
eukaryotic homologs of yeast Cbf5p are shown. The positions of alanine
substitutions introduced at conserved residues in yeast Cbf5p are
indicated by the arrows.
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|
The mutated
cbf5D65A,
cbf5P67A,
cbf5L94A, and
cbf5D95A genes were introduced into
a
cbf5
yeast strain by using a plasmid-shuffle
technique
as described in Materials and Methods. Briefly, in vitro
mutagenesis
was carried out on the
CBF5 gene cloned in pBluescript
KS(

) in
E. coli, and the mutated genes were subcloned into
the
yeast expression vector pBFG, in frame with three HA-epitope tags
and under control of the
PGK1 promoter. The resulting
plasmids
were transferred into yeast strain YCC131, which contains a
wild-type
copy of the
CBF5 gene on the
pYHY18-
CBF5 plasmid covering a chromosomal
deletion of the
essential
CBF5 gene
(
cbf5::
HIS3) (
21). Cells
harboring both the mutant and the wild-type plasmids were plated
for
two rounds on medium containing FOA to select for loss of
pYHY18-
CBF5 (a
URA3-bearing plasmid). In all
cases, FOA-resistant
colonies were obtained (confirmed to be
Leu
+ and Ura

), indicating that all of the
mutant
cbf5 strains are viable in
the absence of a wild-type
copy of
CBF5. The presence of the mutated
cbf5
gene in these yeast strains was confirmed by rescue of the
plasmids in
E. coli and restriction enzyme analysis, since the
mutations
all either created or destroyed restriction
sites.
The mutant strains were checked for their growth properties in a rich
medium (YPD) at various temperatures. The
cbf5D65A,
cbf5P67A, and
cbf5L94A mutations had minimal
effect on growth
at 30°C; however, strain
cbf5D95A had an
unusual slow-growth phenotype
at both 30 and 25°C (Fig.
2A and B). Interestingly, all of the
mutants are temperature sensitive at 37°C but exhibit different
degrees of phenotypic lag. In liquid cultures, strains
cbf5D65A
and
cbf5L94A continue to grow after
shiftup to 37°C at a rate
somewhat reduced from that of the wild
type, with only a brief
phenotypic lag (ca. 2 h) (Fig.
2A). In
contrast, strain
cbf5P67A
grows slower than wild type from
the onset of the temperature
shift and then slows markedly after 3 to
4 h, showing very little
further growth after 8 to 9 h. This
type of slow or delayed-arrest
phenotype is often associated with
mutations resulting in defects
in rRNA transcription or in ribosome
assembly (
34). A similar
slow-arrest phenotype at 38°C was
previously observed with the
temperature-sensitive
cbf5-1
mutant strain, in which rRNA transcription
is severely reduced at the
nonpermissive temperature (
9). Strain
cbf5D95A
did not grow at 37°C, either in suspension or on solid
medium (Fig.
2). All of the mutants exhibited a cold-sensitive
phenotype at 17°C
(Fig.
2B), with
cbf5D95A again showing the most
dramatic
phenotype.

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FIG. 2.
Yeast cbf5 mutants altered by single amino
acid substitutions in the putative synthase catalytic domain have
growth defects. (A) Growth curves of cbf5 mutant and
wild-type strains at 30 and 37°C. Cells were grown overnight at
30°C in liquid YPD to an OD600 of ~0.1. Cultures were
either maintained at 30°C or shifted to the restrictive temperature
(37°C) to monitor the growth rates over a period of 20 h. (B)
The cbf5 mutant and wild-type strains were checked for their
growth properties on solid medium at 17, 25, 30, and 37°C after
incubation for 4 days. In addition to the heat-sensitive phenotype at
37°C, the mutant strains also exhibited cold-sensitive phenotypes
(17°C). (C) Mutationally altered Cbf5p's are expressed in yeast and
accumulate in vivo. Triple HA-tagged wild-type (WT) or mutated Cbf5p's
were expressed from the pBFG vector in a cbf5-null
background. Whole-cell extracts were prepared, and aliquots (25 µg
total protein) were analyzed on Western blots with monoclonal antibody
directed against the triple HA-epitope tag (see Materials and Methods).
As negative control, extract from strain YXCC12-2 containing the pBFG
vector gave no signal with the anti-HA antibody (leftmost lane). The
position of the expected Cbf5p band is indicated.
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|
To determine whether the observed growth defects were due to Cbf5p
instability in the mutant strains, we examined the intracellular
levels
of altered
cbf5 proteins by Western blotting. Whole-cell
extracts were prepared from the wild-type (YCC133),
cbf5D65A,
cbf5P67A, and
cbf5L94A
strains grown for 10 h after a shift to
37°C and from the
cbf5D95A strain grown at 30°C. Aliquots containing
equal
amounts of total protein were analyzed by immunoblotting
with
monoclonal antibody HA.11 directed against the triple HA-epitope
tag.
As shown in Fig.
2C, all of the mutationally altered proteins
were
expressed in vivo, indicating that the growth alterations
of the mutant
strains were due to inherent functional
defect(s).
Pseudouridylation of rRNA is reduced or eliminated in the
cbf5 mutants.
If Cbf5p is indeed the
synthase for
rRNA, alanine substitutions within the putative
synthase sequence
domains should result in reduced
levels in both 18S and 25S rRNA.
We determined the
content of rRNA in CBF5 wild type and
in the cbf5D65A, cbf5P67A, cbf5L94A,
and cbf5D95A mutant strains. De novo-synthesized rRNA was
labeled in vivo with [32P]orthophosphate at both 30 and
37°C, the labeled 18S and 25S rRNA species were gel purified and
subjected to RNase T2 digestion, and the nucleotide
compositions were analyzed by two-dimensional TLC. The
p content of
each rRNA was compared with the Up content of the same RNA species to
obtain a
p/Up ratio. The results from analysis of 18S and 25S rRNA
samples are presented in Table 1 and Fig.
3. In cells grown at 30°C,
contents
of both 18S and 25S rRNA were sharply reduced in mutants
cbf5D65A, cbf5L94A, and cbf5D95A. In
fact, no
could be detected in rRNAs isolated from strain
cbf5D95A under conditions that would permit detection of less than one
per RNA molecule. This result is consistent with the
absolute requirement of this aspartate (D95) in the active site of a
known prokaryotic
synthase (20). When the labeling was
carried out after a shiftup to 37°C,
contents in mutants D65A and
L94A were reduced even further. Thus, cbf5L94A 18S and 25S
rRNA
levels were reduced to 6 and 5% of wild-type levels, respectively, when cells were labeled 1.5 h after shiftup and were
below the limit of detection when labeled 10 h after shiftup (Table 1). However, in mutant cbf5P67A cells grown either at 30 or 37°C, rRNA
levels were found to be consistently above the
levels observed in our CBF5 wild-type strain, although it is
not certain that the observed differences are significant.

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FIG. 3.
Mutations in the conserved synthase domains of Cbf5p
inhibit pseudouridylation of rRNA. In vivo 32P-labeled 25S
rRNAs from wild-type or cbf5 mutant cells were extracted
from agarose-formaldehyde gel slices, digested with RNase T2, and
subjected to two-dimensional TLC (see Materials and Methods). Labeled
spots corresponding to Ap, Gp, Cp, Up, and p are indicated in the
diagram. The various panels show phosphorimages of TLC-fractionated 25S
rRNA hydrolysates prepared from the following strains. (A) YCC37, a
wild-type CBF5 expressed from pFAT10 (left panel); YCC35, a
Drosophila homolog of CBF5; and Nop60B, expressed
from pADNS (right panel). (B) YPH274, a CBF5/CBF5 wild-type
isogenic control strain (left) and YWJ64-ts, cbf5-1/cbf5-1
(right) (9). (C) YCC133, a wild-type CBF5
covering the cbf5-null allele, grown at 30°C (left) or
37°C (middle) for 10 h or at 37°C for 1.5 h (right). (D
to F) Strains harboring cbf5 mutations D65A, P67A, and L94A,
respectively, covering the cbf5-null allele, grown at the
permissive temperature 30°C (left) or at the restrictive temperature
37°C for 10 h (middle) or 1.5 h (right). (G) Strain
harboring the cbf5D95A point mutation grown at 25°C (left)
or 30°C (right). The patterns shown are from analyses with 17,000 cpm
of digested RNA per TLC plate. Essentially identical results were
obtained with samples containing 100,000 cpm per plate, which increased
the sensitivity of detection to less than one residue per RNA
molecule.
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With the obvious exception of mutant
cbf5P67A, there is a
rough correlation between the degree of

depletion and the severity
of the growth defects seen in these mutants. For example, strain
cbf5D95A shows the most severe growth defect (very slow
growth
at 30°C and no growth at 37°C) and contains no detectable

in
its rRNA. Strains
cbf5D65A and
cbf5L94A
have pronounced temperature-sensitive
growth phenotypes and contain
markedly reduced levels of

at
higher temperatures. Except for the
physiological defect associated
with the absence of the
rluD

synthase in
E. coli (
43), we
know of no other
reports that suggest a phenotype associated with
the loss of

in
rRNA. However, we cannot rule out the possibility
that the growth
defects associated with these mutations are due
to

-independent
functions of Cbf5p (see
below).
The temperature-sensitive
cbf5-1 mutant was recently shown
to have a defect in rRNA transcription and a reduced level of 40S
and
60S cytoplasmic ribosomal subunits at the nonpermissive temperature
38°C (
9). Surprisingly, analysis of

content of 18S
rRNA (data
not shown) and 25S rRNA from a
cbf5-1 strain
(YWJ64-ts) labeled
after incubation for 10 h at the nonpermissive
temperature revealed
normal

levels (Table
1 and Fig.
3), findings
similar to those
observed with an isogenic
CBF5 wild type
(YPH274) and mutant
cbf5P67A.
Thus,
cbf5-1 and
cbf5P67A are examples of Cbf5p structural alterations
resulting in a temperature-sensitive growth phenotype in the absence
of
any effect on

formation in rRNA. The position(s) of amino
acid
substitution(s) in
cbf5-1 is
unknown.
When expressed in yeast, Nop60B, the
Drosophila homolog of
CBF5, was recently shown to partially complement the lethal
cbf5::
HIS3 null allele, while the rat
homolog Nap57 failed to do so (
42).
Growth of a haploid
cbf5::
HIS3 strain, harboring the fly
Nop60B
cDNA expressed from the
ADH1 promoter on a 2-µm
plasmid, is considerably
slower than that of a
CBF5
wild-type strain. Nop60B has 63% identity
over 380 amino acids to
Cbf5p. Although the N-terminal and the
C-terminal ends vary in Nop60B,
the putative

synthase domain
is highly conserved between the two
proteins (Fig.
1B). The
content of rRNA in this strain (YCC35) was
examined and, surprisingly,
YCC35 is also devoid of

in 25S rRNA
(Table
1 and Fig.
3A, right
panel) or 18S rRNA (data not shown). The
severe growth defect
seen in YCC35 is quite similar to that of mutant
cbf5D95A, which
also lacks

in rRNA. However, as with
cbf5D95A, it is unclear
whether the slow-growth defect of
YCC35 is due entirely to loss
of

in rRNA or reflects a weak
functional complementation of
some other Cbf5p
function.
The growth defect correlating with
depletion is not relieved by
Pol II-driven transcription of rRNA.
We have shown previously that
the observed rRNA transcriptional defect in the cbf5-1
conditional mutant can be partially suppressed by expression of rDNA
from the Pol II-driven Gal7 promoter on pNOY103
(9). Because rRNA
content is normal in
cbf5-1, Cbf5p must play an essential role in Pol I-driven
rRNA transcription in addition to its
synthase function. Therefore,
it was important to determine whether growth defects of the mutant
strains, particularly strains cbf5L94A and
cbf5D95A that completely lack
in rRNA, are also
suppressed by pNOY103. To this end, we transformed pNOY103 into each
mutant strain, and individual transformant colonies were inoculated
into glucose (repressed condition) or galactose (induced expression of
rDNA) medium to measure relative growth rates. The mutants fell into
two classes with respect to suppression by pNOY103 (Table
2). The doubling times of the wild-type,
cbf5D65A, and cbf5L94A strains (all containing
pNOY103) were identical under repressed or nonrepressed conditions.
Interestingly, strain cbf5D95A/pNOY103, which grows slowly,
with a doubling time of 4.5 h in glucose, fails to grow upon
transfer to galactose medium. However, mutant cbf5P67A/pNOY103 has a doubling time of 3.5 h in
glucose and 3.0 h in galactose; thus, the expression of rRNA from
a Pol II promoter partially relieves the growth defect of this mutant
at 37°C. A similar result was obtained for the
cbf5-1/pNOY103 strain (YHY64
1/pNOY103) grown in galactose
medium, a result consistent with the suppression results reported
previously (9). Class I mutants, which include cbf5D65A, cbf5L94A, and cbf5D95A, are
not suppressed by pNOY103, indicating that the growth defect associated
with reduction or loss of
in rRNA in these strains is not due to a
Pol I-related transcriptional block. The growth defect in class II
mutants cbf5P67A and cbf5-1 is partially
suppressed by pNOY103, suggesting the presence of a Pol I-specific
transcriptional defect in these strains.
Analysis of pre-rRNA processing in the cbf5 mutant
strains.
It has been proposed that the majority of
pseudouridylation in rRNA occurs on the 35S pre-rRNA prior to the exo-
and endonucleolytic cleavage events that yield mature 18S, 5.8S, and
25S rRNA species (6). There is evidence for the presence of
at least 30 to 35
residues in 35S rRNA, suggesting a potential role
for pseudouridines in controlling pre-rRNA processing (40).
The availability of cbf5 mutants with reduced or completely
depleted
in rRNA should allow us to assess the relationship of
pseudouridylation and pre-rRNA processing.
Pulse-chase rRNA labeling experiments were used to analyze the
processing of newly synthesized rRNA in the
cbf5 mutant
strains
at permissive and nonpermissive temperatures. Wild-type and
mutant
strains were grown to mid-log phase at 30°C and transferred to
37°C for 2 or 10 h (with the exception of
cbf5D95A,
which was
maintained at 30°C) prior to pulse-labeling with
[
3H]uracil. After a chase with an excess of unlabeled
uracil, total
RNAs isolated from aliquots containing equal numbers of
cells
were fractionated on 1.2% agarose-formaldehyde gels and
visualized
by fluorography as described in Materials and Methods. The
processing
of pre-rRNA in strains
cbf5D65A,
cbf5P67A, and
cbf5L94A shifted
to 37°C for 2 or
10 h before labeling was similar to that of the
wild-type strain
(Fig.
4A and B). The major processing
intermediates,
35S, 27S, and 20S pre-rRNAs, were visible in all strains
1 min
after initiation of the chase, but were processed at essentially
normal rates. There did appear to be some low-level accumulation
of
these precursors in mutant D65A (Fig.
4A). However, both the
18S and
25S mature species in these three mutants were synthesized
at rates
almost identical to that of the wild-type strain. The
normalized
25S/18S ratios for rRNA samples isolated from cells
pulse-labeled
10 h after the shiftup to 37°C (20 min chase) were
as follows:
P67A, 1.09; D65A, 1.03; L94A, 1.03; and
CBF5 wild-type,
1.00. Mutant
cbf5P67A incorporated comparatively less
[
3H]uracil into the newly synthesized rRNA species when
labeled
after growth for 10 h at 37°C (Fig.
4A), although rRNA
synthesis
was relatively normal after only 2 h at 37°C (25S/18S
ratio of
1.02) (Fig.
4B). This reduced synthesis of rRNA in
cbf5P67A coincides
with the marked reduction in growth rate
of this strain occurring
7 to 8 h after the temperature shift
(Fig.
2A). This is expected
since rRNA biosynthesis is closely coupled
to the growth rate
of cells (
34). However, there is no
evidence for significant
accumulation of pre-rRNA precursors in this
strain.

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FIG. 4.
Pulse-chase labeling of rRNA in the cbf5
mutant strains. Wild-type or cbf5 mutant cells were pulsed
with [3H]uracil for 3 min at the permissive temperature
or after shift to the nonpermissive temperature and then chased with
excess cold uracil for 1, 5, 10, or 20 min as indicated. See Materials
and Methods for details. Cultures were preincubated for 10 h at
37°C (A) or for 2 h at 37°C (B) prior to pulse labeling. (C)
Wild-type or mutant cbf5D95A cells were grown 48 h at
30°C (to an OD600 of 0.480) before pulse-labeling (3 min)
at the same temperature. The expected positions of the various
pre-rRNAs and mature 18S and 25S RNAs are indicated.
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Pre-rRNA processing was examined in the
cbf5D95A mutant
cells by pulse-labeling after growth for 48 h at 30°C. Although
total
RNA was isolated from equal numbers of cells,
cbf5D95A
contained
a reduced amount of 18S and 25S rRNA species compared to wild
type on ethidium-stained gels (data not shown). Similarly, the
net
incorporation of [
3H]uracil into newly synthesized rRNA
is substantially reduced
in this strain (Fig.
4C). The major 35S, 27S
and 20S processing
intermediates were visible 1 min after initiation of
the chase
and were processed at normal rates. Interestingly, the ratio
of
newly synthesized 25S to 18S rRNA is increased to approximately
1.5 (normalized to wild-type
CBF5 at 1.0), suggesting a strong
defect in 18S accumulation in the D95A mutant (Fig.
4C). However,
the
relatively small amount of 20S pre-rRNA visible on the blots
appears to
be normally processed to mature 18S rRNA. Furthermore,
the newly
synthesized 18S rRNA does not appear to be less stable
than wild type,
since it accumulated gradually during the 20-min
chase. This reduction
in the overall synthesis of rRNA in strain
cbf5D95A is not
improved by supplying 35S precursors transcribed
from a Pol II promoter
(see
above).
It has been reported that genetic depletion of the RNA or protein
components of snR30 (
35) or snR10 (
50) inhibits
processing
of the 35S large pre-rRNA to yield the 20S pre-rRNA (the
immediate
precursor to 18S), thereby preventing the synthesis of mature
18S rRNA. This inhibition leads to accumulation of an aberrant
23S
precursor. Thus, depletion of Cbf5p or of snR30 RNA results
in
defective synthesis of 18S rRNA (
25,
35). To investigate
this possibility in the
cbf5 mutants, steady-state levels of
precursor
and mature rRNA species were examined by Northern blot
hybridization
with selected hybridization probes to various pre-rRNAs,
as well
as to mature 18S and 25S rRNAs. The hybridization probes we
used
are indicated in Fig.
5A. A probe
specific to the internal transcribed
spacer 1 (ITS1) was used to detect
the 20S rRNA, the immediate
precursor to 18S rRNA, while a probe
complementary to the 5' region
of internal transcribed spacer 2 (ITS2)
was used to detect 27S
RNA, the precursor to 25S rRNA. Probes specific
to 18S and 25S
rRNAs were used to detect the mature species. The 35S
rRNA is
detected by all of the probes. The blots reveal a low-level
accumulation
of the 20S and 27S precursors in P67A and L94A (Fig.
5C
and D),
although the increase over wild-type levels is not dramatic. As
expected,
cbf5D95A cells contained considerably less total
rRNA
than did the wild type. However, no abnormal accumulation of
precursors
was apparent; the pre-rRNAs were faintly visible and
corresponded
to the expected positions of these intermediates in the
wild-type
strain (data not shown). Both 18S and 25S rRNAs were detected
with probes to the mature rRNA species and, as predicted by the
pulse-labeling experiments, D95A cells were found to be relatively
deficient in 18S rRNA (Fig.
5B). We could not detect the aberrant
23S
precursor previously shown to accumulate instead of 20S rRNA
upon
depletion of Cbf5p or those snoRNAs essential for processing
of
precursors to 18S rRNA (
25). We conclude that none of the
cbf5 mutants, even those completely lacking

in rRNA,
dramatically
accumulate any major processing intermediates under these
experimental
conditions. Also, there is no direct correlation between
loss
of

in rRNA and defective synthesis of 18S rRNA, since
cbf5L94A,
which completely lacks

in rRNA synthesized at
37°C, contains
normal levels of 18S rRNA. We cannot eliminate the
possibility
that
cbf5L94A cells contain a low, undetectable
level of

at
positions critical for 18S rRNA maturation. However, it
has been
recently demonstrated that, consistent with our own
observations,
loss of function of Gar1p, another snoRNP-associated
protein,
results in inhibition of

formation in rRNA independent of
effects
on 18S rRNA processing (
8).

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FIG. 5.
Steady-state levels of pre-rRNAs and mature rRNA species
in wild-type and selected cbf5 mutant strains. The indicated
yeast strains were grown in YPD at 30°C or 37°C to identical cell
densities. RNA was extracted from approximately equal numbers of cells,
resolved on 1.2% agarose-formaldehyde gels, and transferred to a nylon
membrane. (A) Schematic showing the various pre-rRNAs and the
hybridization probes used in these experiments. (B) Blots were probed
with labeled oligonucleotides 1 (18 nt) and 4 (19 nt), complementary to
the 5' ends of mature 18S and 25S rRNAs. (C) The same membranes were
stripped and rehybridized with oligonucleotide 2 (17 nt), which was
specific to the 5' region of ITS1 upstream of cleavage site A2. (D) The
same membranes were stripped and reprobed with oligonucleotide 3 (18 nt), specific to the 5' region of ITS2.
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We have also analyzed pre-rRNA processing in strain YCC35
(
cbf5::
HIS3/Nop60B), which also lacks
measurable

in rRNA. Since
YCC35 is Ura
+, cells were
pulse-labeled with [
3H-
methyl]methionine,
which labels the methyl groups on the large
precursor. Analysis of the
newly synthesized rRNA species in YCC35
gave a result quite similar to
that obtained with
cbf5D95A. Substantially
lower levels of
newly synthesized 18S rRNA and its immediate 20S
precursor were
synthesized in this strain compared to that of
the wild-type isogenic
strain, YCC37 (Fig.
6). The small amount
of labeled 20S seen early in the chase was further processed over
time
to 18S rRNA. The level of labeled mature 25S rRNA was also
slightly
less than that seen in the wild-type strain. Both mature
rRNA species
appeared stable during the 20-min chase period. In
terms of the
similarity in slow-growth phenotype, lack of

in
rRNA, and reduced
synthesis of 18S mature rRNA, the defects observed
in YCC35 correlate
well with those of mutant
cbf5D95A.

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FIG. 6.
Strain YCC35, with Nop60B (the Drosophila
homolog of yeast CBF5) covering the cbf5 null,
is defective in synthesis of 18S rRNA. Strain YCC35 and the isogenic
CBF5 wild-type strain YCC37 were grown for 48 h at
30°C prior to the pulse-labeling with
L-[3H-methyl]methionine for 3 min
at 30°C. The cultures were chased with excess unlabeled methionine
for 5, 10, or 20 min. RNA was isolated from equivalent numbers of
cells, separated on 1.2% agarose-formaldehyde gels, and visualized by
fluorography. The expected positions of the various pre- and mature
rRNAs are indicated.
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Mutational alteration of the
synthase domain in Cbf5p can
affect formation of snoRNP complexes.
Results from various
laboratories indicate that Cbf5p, Gar1p, Nhp2p, and Nop10p combine with
various box H/ACA snoRNAs to form the RNP complexes (H/ACA snoRNPs)
required for pseudouridylation of rRNA (8, 19, 25, 58). In
addition, Cbf5p has been shown to associate with all members of the box
H/ACA snoRNAs (25). The growth defects seen in our
cbf5 mutants possibly could result from an inability of the
altered cbf5 proteins to complex properly with the snoRNAs.
We therefore asked whether any of the amino acid substitutions in Cbf5p
altered the physical association of the mutated proteins with the H/ACA
snoRNAs. We utilized a coimmunoprecipitation strategy as described in
Materials and Methods. Briefly, mutant and wild-type strains were grown
overnight at 30°C, and aliquots were transferred to 37°C and grown
for an additional 1.5 h (except cbf5D95A was maintained
at 30°C). Cell lysates were prepared and immunoprecipitated with
monoclonal antibody HA.11 (see Materials and Methods) directed against
the triple HA-tagged Cbf5p. Total RNA was extracted from the
immunoprecipitates, fractionated on denaturing gels, and analyzed on
Northern blots with a mixture of labeled hybridization probes specific
for four H/ACA snoRNAs: snR8, snR10, snR31, and snR37. In mutant L94A
and P67A cells kept either at 30 or 37°C for 1.5 h, snoRNP
particle content is at wild-type levels, indicating that the physical
association of these mutated Cbf5ps with the snoRNAs is relatively
normal (Fig. 7). However, mutant D65A
cell extracts, prepared from cells grown at either temperature, contain
decreased quantities of the four analyzed snoRNAs (Fig. 7, lanes marked
D65A [T]); although most of these snoRNAs appear to be associated
with the immunoprecipitated Cbf5p-containing snoRNP particles (Fig. 7,
lanes marked D65A [P]). Similarly, extracts prepared from D95A cells
grown at 30°C are quite deficient in both total snoRNAs and snoRNP
particles. Thus, the synthesis and/or the stability of snoRNAs is
impaired in both cbf5D65A and cbf5D95A. The
latter results confirm and extend previously reported observations
indicating that Cbf5p is required for stable association of snoRNAs in
the H/ACA snoRNP complexes (25, 58). It is clear, however,
that the observed severe impairment of
formation in mutant L94A
rRNA is not due to lack of intact snoRNP particles, since snoRNP
content is relatively normal in this strain (at least for the four
snoRNAs analyzed) at 30°C and after 1.5 h at 37°C. Prolonged
(10 h) incubation of the cbf5 mutant strains at high
temperatures does cause an overall reduction in snoRNP particle content
(data not shown), possibly due to secondary effects or inherent
instability of the altered snoRNP particles.

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FIG. 7.
Association of HA-epitope-tagged mutant cbf5
proteins with selected box H/ACA snoRNAs. Yeast extracts were prepared
from cells expressing wild-type or mutationally altered cbf5
proteins after growth at the permissive (30°C) or the nonpermissive
(37°C) temperature for 1.5 h. Immunoprecipitations were carried
out with monoclonal antibody directed against the triple HA-epitope
tags. RNA was purified from equal volumes of total extracts (T) or
immunoprecipitated beads (P) and analyzed on 8% acrylamide-7 M urea
gels. RNA was transferred to a nylon membrane by electrophoresis, fixed
by UV irradiation, and probed with a mixture of
32P-end-labeled snR8, snR10, snR31, and snR37
oligonucleotides. For details, see Materials and Methods. The expected
positions of these snoRNAs are indicated.
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Cytoplasmic ribosomal subunit profiles in the cbf5
mutant strains.
Defects in ribosome assembly and/or transport can
be detected by measuring the levels of mature ribosomal subunits in the cytoplasm. Cell extracts were prepared from cbf5 mutant
strains and wild-type cells after incubation for 2 h at the
nonpermissive temperature (for strain cbf5D95A, after
overnight growth at 30°C). Ribosome profiles were obtained by
sedimentation of cell extracts through sucrose gradients under
conditions leading to dissociation of intact ribosomes to the 40S and
60S subunits (see Materials and Methods). Mutant cbf5D65A,
cbf5P67A and cbf5L94A cells contain approximately
wild-type levels of the ribosomal subunits after 2 h at 37°C, as
judged by the relative ratios of the 40S and 60S subunit peaks (Fig.
8A). However, the ribosomal subunit
profiles were severely altered in extracts prepared from
cbf5D95A cells. The level of the 60S ribosomal subunit peak
is severely reduced, and the 40S peak is almost nonexistent in this
mutant (Fig. 8B). Also, the relative positions of the two subunit peaks
are altered in D95A extracts, suggesting the presence of abnormal
particles, such as subunit precursors or degradation products. As
expected from the rather severe effects on rRNA synthesis seen in the
pulse-labeling experiments (Fig. 4C), the D95A mutation also has a
dramatic effect on the production of mature cytoplasmic ribosomes.

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FIG. 8.
Sedimentation profiles of 40S and 60S cytoplasmic
ribosomal subunits from cells expressing wild-type or mutated
cbf5 genes. Exponentially growing cells (30°C) were either
shifted to 37°C for 2 h (A) or maintained at 30°C (B) prior to
harvesting. Approximately 20 OD260 U of each cell extract
was sedimented through a 7 to 47% linear sucrose gradient under
conditions that dissociate ribosomes into the 40S and 60S subunits,
which were analyzed as described in Materials and Methods.
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DISCUSSION |
In yeast cells the products of two genes, PUS4 and
CBF5, have considerable sequence homology to truBp, the
pseudouridine synthase that catalyzes the formation of
55 in
E. coli tRNA (38). Recently, Pus4p was shown to
be the yeast counterpart of E. coli truBp, catalyzing the
formation of
55 in both mitochondrial and cytoplasmic tRNAs
(4). Therefore, it is reasonable to expect that Cbf5p should
also have pseudouridylation activity. Moreover, results from various
laboratories have provided evidence that Cbf5p, together with Gar1p,
Nhp2p, and Nop10p, are the core protein components of the H/ACA snoRNPs
(19, 25, 28, 58). The majority of H/ACA snoRNPs, by virtue
of their association with guide RNAs, are implicated in directing
site-specific pseudouridylation of rRNA (12, 36, 40, 44).
Depletion in yeast of individual H/ACA snoRNP proteins reduces
levels in rRNA, suggesting that a
synthase activity is an integral
component of these snoRNPs. Among these essential snoRNP proteins, only
Cbf5p shares sequence similarity with known or putative
synthases.
In the present study, we provide further evidence indicating that Cbf5p
is the
synthase component of the H/ACA snoRNPs. Substitution of
certain highly conserved amino acids in the putative
synthase
domains of Cbf5p greatly reduces
levels in rRNA. In fact,
alteration in Cbf5p of D95 in the XLD motif, previously shown to be
essential for the catalytic activity of truAp, an E. coli
tRNA
synthase (20), appears to result in complete loss
of
in yeast rRNA (Table 1). However, two of these mutations (D65A
and D95A) result in reduction of both rRNA
content and snoRNP
levels. In these strains, it is possible that the impairment of
synthesis could be due to the lack of intact snoRNP particles. However,
mutant cbf5L94A shows nearly complete loss of
in both
18S and 25S rRNAs, with no observable reduction of cellular snoRNP
levels (Table 1 and Fig. 7). The most reasonable explanation of these
results is that Cbf5p indeed is the
synthase component of the
snoRNP particles.
All of the cbf5 mutants examined in this study showed some
degree of growth impairment, especially at high temperatures (Fig. 2).
In some of these mutants, a rough correlation is seen between the
severity of these growth defects and the amount of
depletion in
rRNA. Strains cbf5D65A and cbf5L94A are
temperature sensitive both for growth and for insertion of
in rRNA.
Mutant cbf5D95A, which lacks
in rRNA even at permissive
temperatures, shows the most severe growth defect. These results
suggest that pseudouridylation of rRNA may be required for normal
growth of yeast cells. However, strain cbf5P67A and the
cbf5-1 conditional mutant exhibit temperature-sensitive growth phenotypes in the absence of any effect on
formation. Clearly, Cbf5p and the snoRNPs are involved in functions other than
formation, and thus we cannot exclude the possibility that the
slow-growth defect observed in these cbf5 mutants could be due to inactivation of non-
-related function(s). Recently, it has
been shown that RluDp, an E. coli
synthase required for formation of the universally conserved
1915 and
1917
modifications in rRNA, is essential for normal growth; the absence of
RluDp results in severe growth defects in E. coli
(43). However, no growth defects have been previously
associated with loss of function of other known rRNA
synthases.
Expression of rRNA from a Pol II-dependent promoter in pNOY103 was
previously shown to suppress the temperature-sensitive growth phenotype
associated with defects in rRNA synthesis in the cbf5-1
conditional mutant (9). However, mutations resulting in
depletion in mutants cbf5D65A, cbf5L94A, and
cbf5D95A were not suppressed by the presence of pNOY103,
indicating that the growth defects associated with
depletion do not
result from transcriptional problems. In fact,
cbf5D95A/pNOY103, which contains no
in rRNA, does not
grow upon transfer to galactose medium, possibly because expression of
rRNA from a Pol II promoter increases the intracellular pool of
undermodified rRNA which might be toxic to cells. In contrast, the
cbf5P67A mutation is partially suppressed by pNOY103,
indicating that this mutant may have a transcriptional defect analogous
to that observed in cbf5-1. Alteration of a highly conserved
proline in the
synthase domain could result in significant conformational changes in Cbf5p, since proline is known to occur at
bends in the peptide backbone (7). The exact mechanism by which Cbf5p exerts its effects upon Pol I-dependent rRNA transcription is still unknown.
The majority of
has been shown to be inserted in the large pre-rRNA
precursor in both prokaryotic and eukaryotic rRNA prior to nucleolytic
cleavages, suggesting a possible role of this modification in
processing (6). Recently, it has been demonstrated that a
conditional mutation (gar1.1) in Gar1p results in inhibition of 18S rRNA production and depletion of ribosomal pseudouridines at the
nonpermissive temperature (8). No direct correlation was
observed between rRNA processing and rRNA pseudouridylation, however,
since depletion of snR30 or U3 similarly prevented 18S rRNA processing
even though
content in the 35S pre-rRNA was normal (8).
Our results indicate that lack of
in rRNA does not result in
dramatic accumulation of precursors, since the
-depleted strains
cbf5L94A and cbf5D65A, rRNA processing is
relatively normal compared to that of the wild-type strain. However, as
with gar1.1, a severe reduction in net synthesis of mature
18S rRNA is seen in cbf5D95A and in the cbf5
strain expressing the Drosophila Cbf5p homology (YCC35),
both of which lack
in rRNA. No processing intermediates were seen
to accumulate in these strains; the residual 20S pre-rRNA, the
immediate precursor to 18S, was processed to mature 18S during a 20-min
chase period. In a Cbf5p-depleted strain, the defect in synthesis of
18S rRNA was previously shown to be accompanied by accumulation of an
aberrant 23S RNA (25). However, we have not detected this
intermediate in strain cbf5D95A. The basis for the
drastically reduced levels of both 18S and 25S rRNAs in this mutant is
still unclear, although it seems likely that rRNAs lacking proper
modifications might be intrinsically unstable in vivo. For example,
could stabilize the secondary structure of rRNA and facilitate
interaction with ribosomal proteins during the assembly process. In
addition, a severe reduction in mature cytoplasmic ribosomal subunits
is seen in cbf5D95A cells (Fig. 8). This is undoubtedly in
large part a direct result of the defect in 18S rRNA biosynthesis. Some
or all of these effects could stem from loss of non-
-related snoRNP
functions, such as chaperoning the folding of pre-rRNA, which in turn
could affect rRNA transcription, processing, modification, assembly of
rRNP complexes, and transport to the cytoplasm.
The conserved H/ACA box elements are required for synthesis and
accumulation of snoRNAs and may serve as binding sites for individual
snoRNP proteins (2, 31, 5). In two of the cbf5 mutants (D65A and D95A), coimmunoprecipitation experiments indicate that both snoRNA and snoRNP levels are severely reduced, a finding consistent with the previous observation that Cbf5p is required for the
stability of H/ACA snoRNAs (25). These results suggest that Cbf5p may also play a role in synthesis and/or accumulation of
this class of RNAs. One possible explanation would be that Cbf5p
directly interacts with the conserved H/ACA boxes to provide metabolic
stability to the snoRNA and to participate in assembly of the snoRNP
particle. Binding to the H/ACA elements could place the catalytic
domain of Cbf5p in direct contact with the substrate uridine, located
invariantly 14 to 16 residues from either the H or the ACA box. Another
interesting possibility is that Cbf5p could be involved in
pseudouridylation of snoRNAs, since it is known certain snoRNAs contain
(17, 30). However, in other
synthase active site
mutants (L94A and P67A), snoRNA and snoRNP contents are normal. Thus,
the relationship between
synthase activity of Cbf5p and the ability
of the protein to participate in the formation of intact snoRNP
particles is still unclear.
Finally, to prove conclusively that Cbf5p functions as a
synthase,
the catalytic activity of this protein must be demonstrated in vitro.
This might be difficult, because the catalytic activity could require
the presence of an intact snoRNP complex. However, the recent
identification of the core protein components of the H/ACA snoRNPs
could eventually lead to the development of an in vitro assay for
formation and further information on the role of
in rRNA structure
and function.
 |
ACKNOWLEDGMENTS |
We thank Mary Baum for technical advice and assistance in
preparing the manuscript, Dottie McLaren for preparing the figures, and
M. Nomura (University of California, Irvine) for providing plasmid pNOY103.
This research was supported by National Institutes of Health research
grants CA11034 from the National Cancer Institute (J.C.), GM33783
(L.C.) and GM19351 (M.J.F.). J.C. is an American Cancer Society
Research Professor.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular, Cellular, and Developmental Biology, University of
California at Santa Barbara, Santa Barbara, CA 93106. Phone: (805)
893-3163. Fax: (805) 893-4724. E-mail:
carbon{at}lifesci.ucsb.edu.
 |
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