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Molecular and Cellular Biology, April 2000, p. 2827-2838, Vol. 20, No. 8
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Role of Nuclear Cap Binding Protein Cbc1p of
Yeast in mRNA Termination and Degradation
Biswadip
Das,1
Zijian
Guo,1,
Patrick
Russo,1,2
Pascal
Chartrand,3 and
Fred
Sherman1,*
Department of Biochemistry and Biophysics,
University of Rochester Medical School, Rochester, New York
146421; Institute for Molecular
Medicine, Albert Einstein College of Medicine, Bronx, New York
104613; and Department of Plant
Pathology, Cornell University, Ithaca, New York
148532
Received 16 September 1999/Returned for modification 20 November
1999/Accepted 16 January 2000
 |
ABSTRACT |
The cyc1-512 mutation in Saccharomyces
cerevisiae causes a 90% reduction in the level of
iso-1-cytochrome c because of the lack of a proper
3'-end-forming signal, resulting in low levels of eight aberrantly long
cyc1-512 mRNAs which differ in length at their 3'
termini. cyc1-512 can be suppressed by deletion of either
of the nonessential genes CBC1 and CBC2, which
encode the CBP80 and CBP20 subunits of the nuclear cap binding complex,
respectively, or by deletion of the nonessential gene UPF1,
which encodes a major component of the mRNA surveillance complex.
The upf1-
deletion suppressed the cyc1-512
defect by diminishing degradation of the longer subset of
cyc1-512 mRNAs, suggesting that downstream elements or
structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512
defects by cbc1-
occurred by two different mechanisms.
The levels of the shorter cyc1-512 transcripts were
enhanced in the cbc1-
mutants by promoting 3'-end
formation at otherwise-weak sites, whereas the levels of the longer
cyc1-512 transcripts, as well as of all mRNAs, were
slightly enhanced by diminishing degradation. Furthermore, cbc1-
greatly suppressed the degradation of mRNAs
and other phenotypes of a rat7-1 strain which is defective
in mRNA export. We suggest that Cbc1p defines a novel degradation
pathway that acts on mRNAs partially retained in nuclei.
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INTRODUCTION |
The lack of normal transcription
termination or 3'-end processing of mRNAs can lead to drastic
reductions in gene expression, as exemplified by the
cyc1-512 mutation of the CYC1 gene, which encodes
iso-1-cytochrome c in Saccharomyces cerevisiae.
The cyc1-512 mutation, consisting of a 38-bp deletion, 8 nucleotides (nt) upstream from the normal poly(A) site, was found to
cause an approximately 90% diminution in the CYC1 mRNA
and in the corresponding iso-1-cytochrome c protein
(88). The cyc1-512 mRNAs were aberrantly
long, with many discrete 3' termini ranging from the wild-type poly(A)
site to endpoints greater than 2,000 nucleotide (nt) downstream.
Apparently, the lack of the normal 3'-end-forming signals resulted in
partial termination or processing at various sites beyond the normal
poly(A) site. The abnormally long mRNAs were suggested to be
rapidly degraded (88).
Genetic analysis revealed the following three types of
cyc1-512 revertants that were isolated on lactate medium, a
medium that requires increased levels of iso-1-cytochrome c
for growth: (i) single- or multiple-base pair changes, resulting in
intragenic revertants that contained new 3'-end-forming signals at
various sites in the 3' untranslated region of CYC1; (ii)
gross chromosomal aberrations that resulted in the formation of
abnormal 3' regions; and (iii) extragenic suppressors that enhanced the
levels of the cyc1-512 mRNA and of iso-1-cytochrome
c by up to approximately fourfold (39, 89). The
cyc1-512 suppressors constituted recessive mutations that
could be assigned to at least two loci, which were designated
SUT1 and SUT2 (39, 89).
As shown in this study, complementation of the suppressors
sut1-2 (now designated cbc1-2) and
sut2-2 (now designated upf1-102) and DNA
sequencing of the appropriate cloned fragments revealed that
SUT1 and SUT2 correspond to the previously
identified genes CBC1 and UPF1, respectively.
CBC1 encodes a protein corresponding to the orthologous
nuclear cap binding protein, CBP80, in animal cells (16, 28,
29), whereas UPF1 encodes one of the major components of the nonsense-mediated mRNA decay (NMD) pathway
(9, 12, 32, 41, 62, 84). Furthermore, the examination of the
abundance and half-lives of cyc1-512 mRNAs and other
normal mRNAs in cbc1-
and upf1-
strains
revealed that Cbc1p may be a component of a novel nuclear mRNA
decay pathway and that mRNAs with extended 3' ends can be degraded
by the NMD pathway. Also, studies with cbc1-
strains
demonstrated that Cbc1p apparently is required to prevent promiscuous
3'-end formation.
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MATERIALS AND METHODS |
Genetic nomenclature.
The wild-type allele encoding
iso-1-cytochrome c is designated CYC1 or
CYC1+. Mutant alleles that produce either normal
or decreased levels of iso-1-cytochrome c are designated
cyc1 followed by the allele number, e.g., cyc1-512.
CBC1 (or CBC1+), for example, denote the
wild-type alleles. The cbc1-2, sut2-2, etc., gene
symbols designate recessive mutant alleles, whereas cbc1-
or cbc1::URA3, for example, denotes a disruptant
of CBC1.
CBC1 was previously designated SUT1 (39,
89), GCR3 (77, 78), and STO1
(11), whereas CBC2 has also been designated MUD13 (11). UPF1 was previously
designated SUT2 (39, 89) before its
identification. As mentioned above, the suppressors sut1-1
and sut1-2, etc., are now designated cbc1-1 and
cbc1-2, etc., whereas the suppressors sut2-1 and
sut2-2, etc., are now designated upf1-101 and
upf1-102, etc.
Strains, media, and yeast genetics.
The strains of S. cerevisiae used in this study are listed in Table 1.
DE3
lysogens of Escherichia coli strain BL21 (F
ompT rB
mB
) (76) was used for the
expression of a portion of Cbc1p. Standard YPD, YPG, SC-Ura (uracil
omission), SC-Leu (leucine omission), and other omission media were
used for yeast propagation and testing (70); chlorolactate
medium was used for the cloning of CBC1 and UPF1
genes (71). Yeast genetic analysis was carried out by
standard procedures described by Sherman (70).
Determination of cytochrome c content.
Total
amounts of cytochrome c were determined by spectroscopic
examination of intact cells at
196°C (71) and by
comparing the intensities of the c C
bands at 547 nm to
strains having known amounts of cytochrome c. More accurate
determinations of cytochrome c content in intact cells were
made by low-temperature (
196°C)-spectrum recordings using a
modified Cary 14 spectrophotometer (25).
Oligonucleotides.
Oligonucleotides used for
oligonucleotide-directed mutagenesis, sequencing, primer extension, and
PCR were synthesized on an Applied Biosystems 380A DNA synthesizer.
Plasmid construction.
Plasmids designated pAB1158, pAB1159,
pAB1160, pAB1733, and pAB1734 were independently isolated from an
S. cerevisiae genomic library constructed in plasmid YCp50
(59) using the complementation strategy outlined below.
pAB1101 was derived from pAB1160 by deleting a 6-kb SalI
fragment and was used to sequence the CBC1 gene. A 4.2-kb
ClaI fragment from pAB1101, containing the CBC1
coding region and flanking sequences, was inserted into pAB625,
resulting in the plasmid pAB1137. The pAB1137 plasmid was used to make
mutations in the CBC1 gene by site-directed mutagenesis.
pAB1135 was constructed by ligating a 4.5-kb
SalI-HindIII fragment from pAB1101 into
YEp51. The plasmid used for disrupting the CBC1 gene,
pAB1100, was constructed by inserting a 2.1-kb
SalI-BamHI fragment, which contained the CBC1 gene, from pAB1101 into the Bluescript SK vector
(Stratagene) and subsequently inserting a 1.1-kb HindIII
fragment, which contained the URA3+ gene from
pYc5, into the HindIII site within the CBC1
gene. pAB81 was derived by inserting a 2.5-kb EcoRI
fragment, containing the cyc1-512 allele, into pBR322.
pAA1735 corresponds to the plasmid YCpPL63 described by Leeds et al.
(42).
The blaster pAB2207 plasmid, used to disrupt
CBC1+, was constructed in three steps.
Initially, a 4.5-kb SalI-HindIII fragment containing the CBC1+ coding region and the
flanking sequence from pAB1101 was inserted into a pUC19 vector. In the
next step, a 1.25-kb BamHI-BglII fragment was
deleted from the CBC1+ coding region and
subsequently replaced by 3.8-kb BamHI-BglII blaster fragment containing a functional yeast
URA3+ gene flanked by 1.1-kb direct repeats of
Salmonella enterica serovar Typhimurium hisG DNA
from pNKY51 (1).
Transformation, nucleic acid isolation, and manipulation.
S. cerevisiae cultures were transformed with plasmid DNA for
complementation analysis and with linear DNA for gene disruption (60) by using the lithium acetate method (27)
followed by selection on SC-Ura (uracil omission) or SC-Leu (leucine
omission) medium. Plasmid DNA was isolated from transformed S. cerevisiae strains as described previously (72). The
yeast chromosomal gene CBC1 was disrupted by transforming
the appropriate yeast strains with DNA fragments that were prepared
either by digesting the plasmid pAB1100 with SalI and
BamHI or by digesting the blaster plasmid pAB2207 with
PvuII and XbaI. Similarly, the UPF1
gene on the chromosome was disrupted by transforming the suitable yeast strains with DNA fragments that were prepared by digesting the plasmid
YCpPL51 (42), with BamHI and EcoRI.
The Escherichia coli strains HB101 and XL1-Blue were
transformed by the protocol of Hanahan (22). Standard
techniques of DNA manipulation, such as cloning, subcloning, and
sequencing, used in this study are described by Sambrook et al.
(67). Nucleic acid and protein sequences were analyzed by
using the computer programs FASTA, TFASTA, BESTFIT, and BLAST from the
University of Wisconsin Genetics Computer Group software version 7.0.
Cloning the CBC1+ and
UPF1+ genes.
The cbc1-2
mutation was originally isolated as a suppressor of cyc1-512
(39, 89). The wild-type CBC1+ gene
was cloned by complementing the cbc1-2 suppressor in strain B-9036 (Table 1) with a YCp50-based yeast
genomic library. Briefly, approximately 105
Ura+ transformants were collected and plated on
chlorolactate medium. Generally, strains with diminished levels of
iso-1-cytochrome c form larger colonies on chlorolactate
medium because of decreased utilization of chlorolactate, which is
toxic (71). Approximately 50 large chlorolactate colonies
were tested for levels of iso-1-cytochrome c by
low-temperature (
196°C) spectroscopic examination of intact cells.
Those strains having diminished levels of iso-1-cytochrome c
were further tested for the levels after loss of the plasmid by
isolating Ura
strains on 5-fluoroorotic acid medium. This
screen resulted in the identification of three strains with lower
levels of iso-1-cytochrome c in a plasmid-dependent manner.
Plasmid DNA was extracted from these three yeast strains, amplified in
E. coli, and shown by restriction endonuclease analysis to
encompass a common region. Furthermore, Northern blot analysis showed
that yeast strains harboring any one of these three plasmids contained
the expected 10% of the CYC1 mRNA, similar to
cyc1-512 strains (data not presented).
DNA sequencing and subsequent computer analysis of the relevant segment
of the complementing plasmid revealed a 2,574-bp open reading frame
(ORF) and the TACTAAC motif upstream of the ORF, which is
indicative of an intron (data not shown). In addition, the putative 5'
and 3' splicing sites could be theoretically assigned by considering
the conserved sequences (86), and these assignments were
confirmed by sequencing the appropriate region of the CBC1 cDNA. The presence of a 322-nt-long intron near the 5' end was also
confirmed by others (73, 78).
The 5' ends of the CBC1 transcripts were mapped by primer
extension to a 60-bp region from
36 to
96 nt. The major
transcription initiation site was located at position
73, flanked by
several minor sites (data not presented). Interestingly, all the 5'
ends aligned at purine residues, and usually at A residues; only the
36 position was at a G residue. The predicted translation product for
CBC1 is 861 amino acids long, a size that would correspond to a protein of 100,017 Da. Comparison of Cbc1p protein with database sequences by using the BLAST method revealed that CBC1 is
identical to GCR3, a gene involved in expression of the
glycolytic genes of yeast (77). However, the initial
published amino acid sequence of GCR3 is not identical to
that of our deduced amino-terminal region because the intron of
GCR3 was not considered by the authors and was included as
part of the ORF.
UPF1 was cloned similarly by the method used to clone
CBC1, except that strain B-9962 (cyc1-512
upf1-102 [sut2-2] ura3-52) (Table 1)
was transformed with the yeast genomic library. Approximately 5 × 105 Ura+ transformants were transferred to
chlorolactate plates, and the 87 largest colonies were chosen for
spectral examination. Four of them were found to have diminished levels
of cytochrome c compared to the parental strain. Plasmids
from these four transformants were isolated and used to transform the
strain B-9962. Two of these four transformants again contained reduced
levels of cytochrome c. Restriction mapping demonstrated
that these two plasmids contained the same insert, which was
approximately 10 kb in size. Approximately 200-nt sequences at both
ends of one insert were determined and compared to the GenBank
database. The results revealed that the insert corresponded to a
segment in chromosome XIII extending from nt 4354 to 13464 of cosmid
9582, accession number Z492559. This region of chromosome XIII contains
the following genes: CHL12 (4522 to 6747), SEC14
(7008 to 8078), UPF1 (8731 to 11646), and MBR3
(12098 to 13114). Because UPF1 is required for the rapid degradation of certain abnormal mRNAs (see below), strain B-9962 was transformed with plasmid pAA1735 (YCpPL63) (42), which
contains the UPF1 gene. Since the level of iso-1-cytochrome
c diminished from 30 to 10% in the sut2-2
transformants bearing the wild-type UPF1+ gene
in plasmid pAA1735, sut2-2 was considered to be complemented by UPF1+, and consequently, SUT2 is
considered to correspond to UPF1. In addition, as described
below, disruption of UPF1 leads to suppression of
cyc1-512.
Analysis of mRNA steady-state levels and stability.
The
stabilities of the various mRNAs and the pre-mRNA were
determined by the inhibition of transcription with 4 µg of
thiolutin/ml at 30°C, as described by Herrick et al. (24).
Total RNA was isolated as described by Russo et al. (65)
from approximately 108 cells, and enriched poly(A) RNA was
isolated from a total of 1 mg of total RNA with the Oligotex mRNA
kit (Qiagen Inc., Valentia, Calif.) as recommended by the vendor.
Northern blot analyses of different mRNAs were conducted as
outlined by Russo et al. (65). Messenger RNA levels were
quantified by storage PhosphorImager analysis (model 425E; Molecular
Dynamics) and normalized against the 18S rRNA signals when total RNA
was used. Signals were normalized against ACT1 mRNA when
poly(A) mRNA was used.
The decay rates and half-lives were estimated with the SigmaPlot
(version 4.0) regression analysis program, using either a single-exponential decay formula, y = 100 e
bx, or a four-parameter double-exponential
decay formula, y = ae
bx + ce
dx (a + c = 100).
The variance was estimated by a nonlinear least-squares regression
analysis using the S-Plus software package, version 3.4 (Mathsoft Inc.,
Cambridge, Mass.).
Antibody production.
An XhoI restriction site was
created 19 bp upstream from the CBC1 3' splicing site in
pAB1137 by site-directed mutagenesis (40). The resulting
XhoI-BamHI fragment, containing 175 codons of
exon 2 of CBC1, was inserted in frame into the expression
vector pET15b (Novagene), resulting in the plasmid pAB1133. The
E. coli expression host BL21 (DE3) was transformed with
pAB1133, and the expression of the target protein was induced by adding
IPTG (isopropyl-
-D-thiogalactopyranoside) to the medium
(76). The 25-kDa Cbc1p fragment, constituting approximately
90% of the inclusion body proteins, was washed twice with washing
buffer (2% Triton X-100, 50 mM Tris-HCl, and 2 mM EDTA, pH 7.5) and
used by Pocono Rabbit Farm (Canadensis, Pa.) to generate rabbit
polyclonal antibodies.
Indirect immunofluorescence and Western blot analysis.
Cells
grown in SC-Leu medium were fixed with 4% formaldehyde at room
temperature for 2 h. Spheroplasts were prepared with zymolase 100T
(ICN Immunobiologicals) and immobilized on polylysine-coated slides
(79). Primary-antibody incubation was performed at 4°C overnight with 10,000-fold-diluted anti-Cbc1p antiserum or preimmune serum. The secondary antibody, Texas red-conjugated goat anti-rabbit immunoglobulin G, was incubated for 30 min at room temperature at a
dilution of 1:100. Nuclei were stained with 1 µg of
4',6-diamidino-2-phenylindole (DAPI)/ml.
Total yeast proteins (14) were separated by sodium dodecyl
sulfate-10% polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose filters. To detect Cbc1p, the
blotted filters were first blocked with phosphate-buffered saline (100 mM NaCl and 100 mM Na2HPO4, pH 7.5) containing
5% nonfat dry milk. After incubation with the primary anti-Cbc1p antiserum at a 1:5,000 dilution for 1 h at room temperature, the blots were washed with a solution of phosphate-buffered saline and
0.1% Tween 20 and incubated with the secondary antibody, horseradish peroxidase-linked donkey anti-rabbit antibody (Amersham), at a dilution
of 1:3,000. The Cbc1p band was subsequently detected with an enhanced
chemiluminescence kit (Amersham).
 |
RESULTS |
Suppression of cyc1-512 by cbc1-
,
cbc2-
, and upf1-
.
The mechanism of
suppression of the cyc1-512 mutation was investigated by
identifying the genes corresponding to the sut1 and
sut2 suppressors. As described in Materials and Methods,
cloning of these two suppressors by complementation and subsequent
sequencing revealed that these suppressors corresponded to
the previously known nonessential genes CBC1 and
UPF1, respectively.
CBC1 encodes the large subunit of the nuclear cap binding
complex (CBC), CBP80, which influences a number of important functions in the maturation and export of capped RNAs (16, 28, 29). CBC1 encodes an 861-amino-acid protein and contains a
322-nt-long intron in the 5' end of the translated region (data not
shown). The yeast Cbc1p is 16.7% identical and 33% similar to the
mammalian CBP80. The other suppressor, UPF1, plays a major
role in the NMD decay pathway (9, 12, 32, 41, 61, 83).
It was originally suggested that the cbc1-2 suppressor coded
for a protein with an altered function which might enhance the 3'-end
formation of the aberrant cyc1-512 transcripts
(89). This hypothesis was tested by disrupting
CBC1 in the haploid strains B-9037 (cyc1-512
CBC1+) and B-9036 (cyc1-512 cbc1-2) (Table
2), which produced 10 and 45% of the
normal level of iso-1-cytochrome c, respectively.
cbc1-2 appeared to be equivalent to a null mutation, since
the phenotypes of the cyc1-512 strains with a disruption of
the CBC1+ gene or with the cbc1-2
allele were identical, having the same amount of iso-1-cytochrome
c (Table 2). Furthermore, cbc1-
did not affect
the expression of CYC1+, a finding which was
consistent with the observation that cbc1-2 did not cause an
increase in the level of iso-1-cytochrome c in a
CYC1+ background (89). In addition,
cbc1-
did not affect the expression of the
cyc1-947 allele that contained a normal 3' untranslated region but that was greatly reduced in transcription due to the lack of
TATA elements (Table 2) (44), suggesting that neither cbc1-2 nor cbc1-
affects the rate of
transcription.
The finding that disruption of CBC1+ had the
same phenotype as cbc1-2, along with the previous
observation that cbc1-2 caused an increase in the
cyc1-512 mRNA level (89), raised the
possibility that cbc1-2 or cbc1-
could act by
increasing the stability of cyc1-512 mRNA.
Therefore, we investigated the stabilities and decay rates of a
number of mRNAs in an isogenic series of strains to test this
hypothesis (see below).
In order to examine whether mutation of either of the components of CBC
could suppress the cyc1-512, we also disrupted the CBC2+ gene (11) in the haploid strain
B-9037 (cyc1-512 CBC2+). Determination of the
level of iso-1-cytochrome c in the resulting B-12463 strain
(cyc1-512 cbc2-
) showed that disruption of
CBC2+ has the same phenotype as that obtained
with the disruption of the CBC1+ gene (Table 2).
Examination of the levels of iso-1-cytochrome c in the
isogenic series of strains listed in Tables 2 and
3 revealed that upf1-
suppresses cyc1-512 to approximately the same extent as cbc1-
and that the double mutant, cbc1-
upf1-
, was not more effective than either of the single mutants
(see Discussion).
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TABLE 3.
Half-lives and relative steady-state amounts of mRNA
and pre-mRNA and steady-state amounts of iso-1-cytochrome
c in cyc1-512 strains having single and
double cbc1- and upf1- mutations
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Disruption of CBC1 or CBC2 diminished cell
growth on glucose medium.
To determine the phenotype of a null
mutant at the CBC1 locus, one copy of CBC1 in a
diploid strain, D-890 (Table 1), was disrupted. Analysis of 11 complete
tetrads from a Ura+ transformant showed that all spores
were viable and that the Ura+ marker segregated 2:2.
Disruption of CBC1 by integration of the URA3
gene was confirmed by PCR amplification of chromosomal DNA isolated
from the Ura+ segregants. Thus, CBC1 is not
essential for cell viability.
Although it was not lethal, disruption of CBC1 or
CBC2 greatly diminished cell growth on glucose medium (YPD),
indicating an important role for Cbc1p in normal cell growth. In
contrast, CBC1+, cbc1-
, and
cbc2-
strains grew nearly the same on nonfermentable glycerol medium (YPG) and fermentable raffinose medium (YPR) (Fig. 1), which extends the early results of
Uemura and Jigmi (77), who reported similar findings with
YPD and YPG.

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FIG. 1.
Comparison of the growth of
CBC1+, cbc1- , and
cbc2- strains. The three isogenic strains were grown at
30°C on YPD glucose medium for 2 days, YPG glycerol medium for 4 days, or YPR raffinose medium for 2 days. (A) B-9037
(CBC1+); (B) B-9038 (cbc1- ); (C)
B-12463 (cbc2- ).
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Cbc1p is located in the nucleus.
The amino-terminal region of
the deduced Cbc1 protein included two basic sequences, NRKRR (residues
3 to 7) and RPRMPKRQR (residues 20 to 28), which could serve as nuclear
localization signals. Although the actual roles of the two basic
regions have not been determined experimentally, their presence at the
amino-terminal region strongly suggests that Cbc1p is localized in the
nucleus. Because of its low abundance in normal cells, Cbc1p could not easily be detected in strain B-6304 by either Western blot or indirect
immunofluorescence analysis. However, Cbc1p became detectable by
overexpression of CBC1 with a 2µm plasmid, pAB1135. Cbc1p, detected by Western blot analysis, was approximately 100 kDa, agreeing
with its predicted molecular mass (data not shown). Furthermore, strain
B-9042, containing pAB1135, was prepared for immunofluorescence microscopic analysis. Experiments with highly diluted anti-Cbc1p antisera demonstrated that Cbc1p was located in the nuclei of the
cells, as was evident from the coincident Texas red and DAPI stains
(data not shown).
To rule out the possibility that Cbc1p could be mislocated to the
nucleus by overexpression, an extract prepared from the normal strain
B-6304 was fractionated into subcellular components (69).
The location of Cbc1p was monitored by protein blot analysis of the
proteins in subcellular fractions. Cbc1p could be readily detected in
the nuclear fraction of strain B-6304 but not in the cytosol fraction
or the total extract (data not shown). Similarly, Görlich et al.
(16) used immunoelectron microscopy to demonstrate that
Cbc1p was located primarily in the nucleus of yeast.
The cyc1-512 mRNAs.
Northern blot analysis
previously revealed a number of cyc1-512 mRNAs,
including 630-, 850-, 1,350-, and 1,450-nt and longer transcripts
(88, 89), whereas 3'-end mapping and sequencing further
defined numerous transcripts between 630 and 700 nt (66). We
have refined the Northern blot analysis and detected the following eight transcripts, as shown in Fig. 2 and
schematically in Fig. 3: 630, 850, 1,450, 1,650, 1,850, 2,000, 2,400, and 3,500 nt. We have designated the 630- and 850-nt mRNAs the short cyc1-512 transcripts and
designated the 1,450- through 3,500-nt mRNAs the long
cyc1-512 transcripts.

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FIG. 2.
Northern blot analyses of the eight cyc1-512
mRNAs listed in Table 3. Poly(A) RNA was extracted from four
strains treated with 4 µg of thiolutin/ml for 0 to 30 min. (A and E)
B-9037 (CBC1+ UPF1+); (B and F)
B-9038 (cbc1- UPF1+); (C and G) B-9848
(CBC1+ upf1- ); (D and H) B-11559
(cbc1- upf1- ). (A to D) cyc1-512 mRNAs;
(E to H) ACT1 mRNA. The values presented in Table 3 were
estimated from these blots by normalizing them to ACT1
mRNA values, whose rates of degradation for these strains are
known.
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FIG. 3.
Schematic representation of the cyc1-512
mRNAs, showing their lengths in nucleotides and the steady-state
increases due to suppression by cbc1- or
upf1- .
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Levels and degradation of mRNAs in cbc1-
and
upf1-
strains.
The following series of isogenic
strains was used for examining the possibility that cbc1-
and upf1-
could act by increasing the stability of
cyc1-512 mRNAs and some other specific mRNAs: B-9037
(cyc1-512), B-9038 (cyc1-512 cbc1-
), B-9848
(cyc1-512 upf1-
), and B-11559 (cyc1-512 cbc1-
upf1-
) (Table 1). The steady-state levels and half-lives of
the following RNAs from these strains were determined by comparing
Northern blots after blocking transcription with the drug
thiolutin (24): the eight cyc1-512
mRNAs, ACT1 mRNA, CYH2 mRNA,
and CYH2 pre-mRNA. 18S rRNA was used as an internal
standard for Northern blots with total-RNA preparations. In one
experiment, where enriched poly(A) mRNA was used to detect the low
levels of the longer cyc1-512 transcripts, ACT1
mRNA was used as an internal loading control, adjusted for the
known rate of decay of ACT1 mRNA in these strains (Table
3). Examples of Northern blots (Fig. 2 and
4), the decay curves (Fig. 5), and the steady-state levels and
half-lives (Table 3) are presented.

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FIG. 4.
Northern blot analysis of CYH2 mRNA and
pre-mRNA (left) from strains B-9037 (CBC1+
UPF1+), B-9038 (cbc1-
UPF1+), B-9848 (CBC1+
upf1- ), and B-11559 (cbc1- upf1- ) treated with
4 µg of thiolutin/ml for 0 to 40 min as indicated above the blots.
18S rRNA is shown on the right-hand side.
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FIG. 5.
Decay in thiolutin-treated cells of cyc1-512
630-nt mRNA (A), CYH2 pre-mRNA (B), CYH2
mRNA (C), and ACT1 mRNA (D) from the following
strains: B-9037 (normal; cyc1-512) ( ); B-9038
(cyc1-512 cbc1- ) ( ); B-9848 (cyc1-512
upf1- ) ( ); and B-11559 (cyc1-512 cbc1-
upf1- ) ( ). The decay was determined by Northern blot
analysis of RNA extracted from cells treated with 4 µg of
thiolutin/ml for 0 to 40 min. The results are presented as the
percentage of remaining RNA versus time of incubation in the presence
of thiolutin.
|
|
The half-lives clearly revealed that the degradation of certain
mRNAs is greatly diminished by the upf1-
mutation. As
expected and as previously observed, the degradation of CYH2
pre-mRNA was greatly diminished in the upf1-
strain
because of the nonsense codon in the CYH2 intron (Fig. 4 and
Table 3) (23), whereas that of CYH2 and
ACT1 mRNAs was not affected (Table 3). Most importantly,
the degradation and steady-state levels of the long cyc1-512 mRNAs were also diminished by two- to
threefold, but not that of the short forms (Fig. 2 and 3 and Table 3).
As discussed below, we suggest that the transcripts longer than 850 nt
contain elements or structures that mimic the signals in mRNAs
having premature terminating codons.
The effect of the cbc1-
mutation was more complex, not
causing diminished degradation of the short (630- and 850-nt)
cyc1-512 mRNAs but causing a pronounced increase in
their steady-state levels (Fig. 3 and Table 3). The stabilities of the
short cyc1-512 mRNAs were the same in all four strains,
but their level are much higher in all cbc1-
strains
(Fig. 2 and 3 and Table 3). This increase is unlikely to be the result
of an enhanced rate of transcription for the following two reasons. (i)
The relative levels of the short and long cyc1-512 mRNAs
were substantially different in the cyc1-512 and
cyc1-512 cbc1-
strains, which would not have been
expected if this increase were due to enhanced transcription. (ii) As
discussed above, cbc1-
did not have any enhancing effect on other CYC1 alleles. These observations suggest that the
increased levels of short (630- and 850-nt) cyc1-512
mRNAs in all cbc1-
strains are due to a phenomenon
designated promiscuous 3'-end formation (see Discussion).
In contrast, the longer cyc1-512 transcripts decayed more
rapidly in the CBC1+ UPF1+ strain
than in the cbc1-
strains, suggesting that degradation of
these longer transcripts was affected by the cbc1-
mutation, similar to the results with the upf1-
mutation
(see above) (Figs. 2A, B, and D). The stabilities, as well as the
steady-state levels, of the longer transcripts increased by at least
two- to threefold in all cbc1-
strains (Fig. 3 and Table
3). Furthermore, cbc1-
also slightly affected the
degradation of other RNAs tested, including ACT1 and
CYH2 mRNA and CYH2 pre-mRNA (Table 3).
Thus, Cbc1p may play a role in the general degradation of all mRNAs.
Upf1-
, but not cbc1-
,
suppresses nonsense mutations.
Both cbc1-
and
upf1-
suppress cyc1-512 by increasing the
levels of one or another of the cyc1-512 mRNAs, but
these suppressors presumably act by different mechanisms. Although both
cbc1-
and upf1-
diminished the
degradation of long cyc1-512 mRNAs, only upf1-
, but not cbc1-
, suppressed
nonsense mutations. This distinction was clearly revealed by
testing the leu2-1 (UAA), met8-1 (UAG), and
his4-166 (UGA) markers (Fig.
6), previously shown to be suppressed by
upf1-
(42). Furthermore, the steady-state
level of the cyc1-72 mRNA, containing a premature UAA
mutation, was greatly enhanced in the upf1-
strain but
not in the cbc1-
strain (Fig.
7).

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FIG. 6.
cbc1- does not suppress the nonsense
mutations. Three strains, B-11623 (UPF1+
CBC1+), B-9198 (UPF1+
cbc1- ), and B-9199 (upf1- CBC1+),
were grown on YPD plates for 2 days, replicated onto omission media
lacking either leucine ( Leu), histidine ( His), or methionine
( Met) and subsequently incubated for 3 days to test suppression of
leu2-1 (UAA), his4-166 (UGA), and
met8-1 (UAG).
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FIG. 7.
Northern blot analysis of B-11623 (cyc1-72),
B-9198 (cyc1-72 cbc1- ), and B-9199 (cyc1-72
upf1- ) showing the CYC1 steady-state mRNA levels
compared to the level of 18S ribosomal DNA (rDNA).
|
|
Suppression of rat7-1 by cbc1-
.
Because
Cbc1p is primarily located in the nucleus (16), we
considered the possibility that the abnormally long cyc1-512 mRNAs are partially retained in the nucleus and are subjected to a
nuclear Cbc1p-dependent degradation system. We have tested this
hypothesis by investigating the decay of specific mRNAs at the
restrictive temperature of 37°C in rat7-1 strains, which
do not grow or export mRNAs under the restricted conditions
(17). All of the rat7-1 strains used in this
study were derived from the same strain previously used by Gorsch
et al. (17). ACT1 and CYH2
mRNAs, as well as CYH2 pre-mRNA, were more
rapidly degraded in the rat7-1 strain than in the isogenic
RAT7+ strain when shifted to 37°C (Fig.
8 and Table
4), although the values of the half-lives
of the CYH2 pre-mRNA in the rat7-1 and rat7-1 upf1-
strains could not be accurately estimated
because of rapid degradation. Furthermore, degradation of all of the
mRNAs tested in rat7-1 strains was suppressed by
cbc1-
but not upf1-
(Fig. 8 and Table 4).
Cbc1-
also suppressed the growth defect inflicted by
rat7-1 at 37°C (Fig. 9). As
discussed below, we suggest that Cbc1p could be required for
degrading mRNAs arrested in the nucleus.

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FIG. 8.
Decay of ACT1 and CYH2 mRNAs
and pre-mRNA at 37°C in B-10603 (RAT7+),
B-10095 (rat7-1), B-10096 (rat7-1 cbc1- ), and
B-10097 (rat7-1 upf1- ). The strains were grown at 23°C
to the mid-log phase, shifted to the restricted temperature of 37°C
for 15 min, and subsequently treated with 4 µg of thiolutin/ml to
inhibit transcription, and the decay was followed for 0 to 50 min.
Northern blots were probed for CYH2 mRNA (bottom band in
each blot) and pre-mRNA (top band in each blot) (A) and for
ACT1 mRNA (B). The numbers at the right denote the
half-lives (t1/2) of the mRNAs in minutes.
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TABLE 4.
Rates of decay of ACT1 and CYH2
mRNAs and pre-mRNA in RAT7+,
rat7-1, rat7-1 cbc1- , and rat7-1
upf1- strains as described in Fig. 8
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FIG. 9.
Growth of B-10603 (RAT7+),
B-10095 (rat7-1), B-10096 (rat7-1 cbc1- ), and
B-10097 (rat7-1 upf1- ) strains on YPD medium at 30 and
37°C for 3 days, showing the suppression of growth in
rat7-1 strains by cbc1- .
|
|
 |
DISCUSSION |
We uncovered three phenomena by systematically investigating two
suppressors that partially restore the diminished levels of
iso-1-cytochrome c and mRNAs in the cyc1-512
mutant that lacks the normal 3'-end-forming signal. First, we observed
that degradation of the cyc1-512 mRNAs longer than 850 nt was suppressed in upf1-
mutants, which lack one of the
main components of the NMD pathway. Second, we found that the long
cyc1-512 mRNAs, and to some extent all mRNAs, are
degraded, presumably in the nucleus, in a Cbc1p-dependent manner.
This degradation is at least partially suppressed by
cbc1-
mutations and presumably is more pronounced
with mRNAs that are retained in the nucleus, either because
of such mutations as rat7-1 or because of structures
of the mRNA, such as abnormal extensions at the 3' end. Finally,
transcription termination and 3'-end formation, which are diminished
because of incomplete or weak signals in the cyc1-512
allele, were found to be enhanced by cbc1-
mutations, a
phenomenon we have designated promiscuous 3'-end formation.
NMD of the long cyc1-512 mRNAs.
The NMD
pathway has evolved as a surveillance mechanism to degrade transcripts
containing premature nonsense codons, thus preventing synthesis of
incomplete and potentially deleterious proteins. In particular, NMD
protects cells from the potentially deleterious effects of inefficient
or inaccurate splicing, which are common and which could result in the
translation of mRNAs with introns. Most introns contain nonsense
codons and are therefore subjected to NMD. Also, certain normal
mRNAs are subjected to NMD, presumably as a means to control
steady-state levels (82). Upf1p is a key component of the
NMD pathway, which exists in all eukaryotes that have been examined
(9, 12, 32, 41, 46, 61, 85).
Muhlrad and Parker (48) and Hagen et al. (21)
demonstrated that NMD proceeds by decapping and subsequent degradation
by the action of the Xrn1p 5'
3' exoribonuclease. Furthermore,
studies of initiation of translation (30, 50) and the
presence of circular polyribosomes in electron micrographs
(31) have suggested that the 5' cap (or 5' untranslated
region) and the poly(A) tail are associated. This interaction between
the 5' cap and the poly(A) tail may prevent decapping (48).
It has been proposed that in-frame premature nonsense codons near the
5' region cause release of the 80S ribosomes and expose an element,
designated the downstream element (55, 62, 63, 93) or the
sensitive element (87), which is situated downstream of the
termination codon. One intriguing model that has been proposed is that
this element may mediate an important interaction between a scanning
ribosome or 40S ribosomal subunit, a consequence of which would be
rapid decapping and degradation by Xrn1p, the major cytoplasmic 5'
3'
exoribonuclease (56, 93). As discussed by Yun and Sherman
(87), 40S ribosomal subunits and RNPs, as well as 80S
ribosomes, may serve to prevent interactions with these hypothetical
downstream elements.
The positions of the termination codons determine whether the NMD will
be triggered, and all of the yeast genes, URA3
(45), URA1 (54), PGK1
(55), HIS4 (21), and CYC1
(87), that have been investigated showed very low abundance
of mRNA when the nonsense mutations were situated at or near the 5'
regions and normal or nearly normal levels when the nonsense mutations
were in the 3' regions of the ORF. This position-dependent triggering
suggests that premature termination before the downstream elements is
required for NMD. It has been suggested that a surveillance complex,
which is composed of Upf1p and other proteins, scans 3' of the
termination codon for these elements. Peltz, Ruiz-Echevarría,
Zhang, and colleagues (55, 62, 63, 93) suggested that rapid
decapping and 5'
3' decay of the mRNA occurs when the
surveillance complex interacts with the downstream element. The
sequence of the downstream element is degenerate and is present
numerous times in virtually all mRNAs (93).
The NMD of cyc1-512 mRNAs can be simply explained by the
presence of downstream elements, or structures in the extended 3' region, specifically between 850 and 1,450 nt, as schematically presented in Fig. 3. In fact, the lack of a downstream element in such
a large segment would be highly unlikely, in view of the fact that all
or most mRNAs with premature nonsense mutations are subjected to
the NMD pathway. Thus, it is reasonable to suggest that the same
mechanism is used for degrading long cyc1-512 mRNAs and
wild-type mRNAs that are targets of NMD. Our discovery of the
involvement of the UPF1 gene in the degradation of long
cyc1-512 mRNAs defines a novel class of substrates of
the NMD pathway, which confirmed similar observations by Muhlrad and
Parker (49). The cyc1-512 type of mRNA with
extended 3' sequences differs from the mRNAs subjected to NMD by
leaky scanning within ORFs (82).
The reduction in mRNA level caused by a mutation or deletion of the
poly(A) signals has also been observed in the r293 mutation of the unc-54 gene in Caenorhabditis elegans,
which involves deletion of the poly(A) signal in the myosin heavy chain
B mRNA (58), and in human thalassemias resulting from
mutations in the conserved AAUAAA polyadenylation signal
(26, 52, 64). In C. elegans, several
smg mutations were shown to suppress the unc-54
(r293) mutation, resulting in near-normal amounts of the
unc-54 (r293) mRNA (8, 58). Two
other deletion mutations that removed the polyadenylation site,
unc-17 (p1156) and unc-17
(md1447), were also suppressed by the smg
suppressors (J. M. Rand, personal communication). One of the
smg genes, smg-2, is homologous to the yeast
UPF1 gene (53), suggesting conservation of a
mechanism which restricts the levels of abnormal mRNAs.
Cbc1p-dependent mRNA degradation.
The identity of
SUT1 and CBC1 established a role of the nuclear
cap binding protein, Cbc1p, in mRNA degradation. CBC is composed of
two proteins designated CBP80 and CBP20 in higher eukaryotes (28,
29, 34, 35, 51) and Cbc1p and Cbc2p, respectively, in yeast.
Mutant forms of CBC1 and CBC2 have been uncovered
in other genetic screens, and CBC1 has been previously
designated not only SUT1 but also GCR3 (77,
78) and STO1 (11), whereas CBC2
has also been designated MUD13 (11). CBC binds to
nuclear RNAs containing the capped 7-methyl-guanosine structure
and, at least in mammalian cells, is involved in pre-RNA splicing,
poly(A) site cleavage, and snRNA export (15, 28, 29, 34, 35, 51). Although CBC translocates through nuclear pore complexes attached to the 5' ends of mRNPs in higher eukaryotes (81), it does not appear to play a direct role in their export. Similar to
higher-eukaryotic CBC, yeast CBC is primarily located in the nucleus
and functions in pre-RNA splicing (11, 43).
The requirement for CBC in pre-RNA splicing was revealed from the
findings that immunodepletion of CBP80 greatly diminishes the activity
in vitro of splicing extracts from higher-eukaryotic cells
(29) and yeast (43). In addition,
cbc2-
strains have diminished pre-mRNA splicing
efficiency in vitro and the cbc2-
mutation is
synthetically lethal with a U1 snRNA mutation (11). Similarly, evidence for the role of CBC in 3'-end formation was obtained by Flaherty et al. (15) using HeLa extracts
with CBP80 immunodepleted. Cleavage of capped pre-RNA was inhibited to
approximately 80%, whereas polyadenylation of a precleaved
substrate was not significantly affected.
Although the primary sequence and the function in splicing of CBC are
conserved from yeast to humans, CBC is not essential for growth in
yeast. Growth of cbc1-
strains is severely retarded on
glucose medium but only mildly diminished on glycerol and raffinose media. The dispensability of CBC in vivo can be explained by only a
partial diminution of splicing and near-normal export of the bulk of
polyadenylated RNAs in cbc1-
strains. Also, the role of
CBC in the export of U snRNAs has not been demonstrated in yeast.
We have presented evidence that Cbc1p is involved in the rapid decay of
long cyc1-512 mRNAs without affecting the decay rates of
shorter 630- and 850-nt transcripts. Interestingly, the degradation of
short cyc1-512 mRNAs followed at least two-component
decay kinetics in a CBC1+ UPF1+
strain, indicative of heterogeneous mRNA molecules having different half-lives (Fig. 5A). We suggest that the long cyc1-512
mRNAs are undergoing rapid degradation in the
CBC1+ UPF1+ strains by Cbc1p- and
Upf1p-dependent degradation systems, and these partially degraded
products of the long forms are situated at the same positions as the
630- and 850-nt bands, thus contributing to the heterogeneous mRNA
molecules having different half-lives.
The major question resulting from this work is the nature of the
mRNA degradation that is dependent on Cbc1p, or presumably on CBC,
since both cbc1-
and cbc2-
equally
suppressed cyc1-512. The decay of longer labile
cyc1-512 mRNAs was diminished in both the
cbc1-
and upf1-
strains, whereas the
degradation of the transcripts containing premature nonsense codons,
such as cyc1-72 mRNA and CYH2 pre-mRNA,
was diminished in only the upf1-
strain. On the other
hand, only the cbc1-
mutation stabilized all mRNAs arrested in the nucleus in the rat7-1 strain and slightly
stabilized all mRNAs in RAT7+ normal strains.
The similarity in the degree of suppression of decay of the long
cyc1-512 mRNAs by single upf1-
and
cbc1-
strains and by the double upf1-
cbc1-
strains (Table 2) could suggest that Cbc1p and Upf1p are
part of the same degradation pathway. In this regard it should be noted
that the mRNA decay function can be separated from the activity
modulating translation termination at nonsense codons (83,
84). For example, a subset of upf1 mutations which
altered the Upf1p helicase region inactivated the decay activity but
did not cause nonsense suppression (83), while another,
different subset of upf1 mutations with alterations in an
amino-terminal region of Upf1p suppressed certain nonsense mutations
without preventing the NMD. Thus, it is reasonable to assume that
deletion of certain components of the NMD pathway would diminish the
decay properties without acting as suppressors. Hence, one could
suggest that cbc1-2 or cbc1-
represents such a
mutation that affects decay but not suppression of nonsense mutation
and Cbc1p is part of the NMD pathway. One could further suggest that
NMD takes place in both the nucleus and the cytoplasm but Cbc1p
functions in this pathway solely in the nucleus. The finding that Upf3p
is a nucleus- and cytosol-shuttling protein in yeast (74)
and that NMD of newly synthesized mRNA copurifies with nuclei of
mammalian cells (90-92) is consistent with this view.
However, the following lines of evidence indicate that NMD occurs in
yeast with transcripts translated on cytoplasmic polysomes: Upf1p,
Upf2p, and Upf3p are associated with polysomes (3, 4, 55,
56); NMD can be suppressed with tRNA nonsense suppressors (5, 18, 45); a cycle of initiation and termination is
required for NMD, and translation reinitiation can prevent activation
of NMD (61, 63, 87, 90); and nonsense codon-containing RNAs lose and regain their decay properties by the addition and removal, respectively, of the translational inhibitor cycloheximide
(90). We favor the view that Cbc1p-dependent degradation is
independent of NMD, because there is no evidence that CBC is associated
with mRNAs during translation on polysomes and because CBC is
primarily situated in the nucleus with only a brief exposure in the
cytoplasm as part of the nuclear transport cycle. Furthermore, the
decay of CYH2 pre-mRNA in rat7-1 strains was
not specifically diminished in cbc1-
strains, which is
one of the natural substrates for NMD (Fig. 8A and Table 4), indicating
that Cbc1p is not involved in the degradation of substrates containing
premature nonsense codons.
Thus, we suggest that Cbc1p-dependent degradation takes place in the
nucleus and may be related to the stimulatory effect of CBC on
pre-mRNA processing, including splicing and cleavage during
3'-end formation. We also argue that all mRNAs located in the
nucleus are degraded by the Cbc1p-dependent pathway and that the net
destruction of a particular mRNA is dependent on the length of time
spent in the nucleus. Even though there is no direct evidence, we
hypothesize that the long cyc1-512 mRNAs are
preferentially retained in the nucleus and we further suggest that this
retention in the nucleus is responsible for their high susceptibility
to the Cbc1p-dependent degradation system.
The hypothesis that Cbc1p is part of a degradation system acting on
mRNAs retained in the nucleus is also supported by the suppression
of rat7-1 by cbc1-
. Rat7p is an essential
nucleoporin 1,450 amino acids long that apparently plays a direct
role in the nucleocytoplasmic export of mRNA (13, 17).
The rat7-1 mutation is a single-base pair change introducing
a premature stop codon approximately 100 amino acids upstream from the
C terminus (13). After rat7-1 cells are shifted
to 37°C, the restrictive temperature, there is a cessation of growth
and of mRNA export, resulting in a diminished level of cytoplasmic
mRNAs. We have observed that the stabilities of
CYH2 mRNA and pre-mRNA, ACT1 mRNA, and presumably other mRNAs were greatly diminished in the rat7-1 mutants grown at 37°C compared to those in a
corresponding isogenic RAT7+ strain (Fig. 8), a
condition that allows growth of the RAT7+
strain. Most importantly, cbc1-
rescues both the
rat7-1 phenotypes, lack of growth (Fig. 9) and instability
of ACT1 and CYH2 mRNAs and CYH2
pre-mRNA at 37°C (Fig. 8).
While the cbc1-
suppression of rat7-1 at
37°C can be interpreted in several ways, including the suppression of
the diminished export, we favor the view that nuclear retention
of mRNAs in rat7-1 strains results in increased
degradation by the action of the Cbc1p-dependent degradation system.
This nuclear degradation leads to diminished levels of mRNAs that
are available for the already abnormally reduced export to the
cytoplasm. In other words, the suppression of rat7-1 by
cbc1-
could be envisioned as enhancing the levels of
nuclear mRNAs and thus allowing the defective transport system to
produce a higher level of cytoplasmic mRNAs required for viability.
Recently, Uemura et al. (78) reported that a cbc1
mutation suppressed the temperature-sensitive growth of hpr1 mutants, which are conditionally defective in the nuclear export of
poly(A) RNA (68). We believe that cbc1
suppression of both hpr1 and rat7-1 may be
operating by the same mechanism. It remains to be seen if
cbc1 suppression of hpr1 and rat7-1 is
due to diminished degradation of mRNAs retained in the nucleus or
to reversal of the mRNA transport defects.
There are a few known ribonucleases that possibly could be components
of the Cbc1p-dependent nuclear degradation system. Two possible
candidates are Rrp6p, which is a nuclear 3'
5' riboexonuclease (6, 7), and Rat1p, which is a nuclear 5'
3'
riboexonuclease (2, 33, 37, 57, 75). Burkard and Butler
(7) suggested that Rrp6p plays a role in a nuclear RNA decay
pathway that destroys slowly processed mRNAs. Furthermore,
two-hybrid analyses and coimmunoprecipitation experiments demonstrated
that Rrp6p interacts with Npl3p and Cbc1p (7;
K. T. Burkard and J. S. Butler, personal communication). Further studies, which are in progress, would reveal the role of Cbc1p
and other components in the nuclear degradation system.
Promiscuous 3'-end formation.
The finding of higher levels of
630- and 850-nt cyc1-512 mRNAs in cbc1-
mutants, without a corresponding increase in the half-lives (Table 3),
suggested that the enhanced levels were due to more efficient 3'-end
formation at these normally weak sites. Extensive work (reviewed in
references 19 and 20) has established that 3'-end-forming signals in yeast consist of three elements: (i) the efficiency element, which enhances the efficiency of
downstream positioning elements; (ii) the positioning element, which
positions the poly(A) site; and (iii) the actual poly(A) site. These
three elements are not only necessary but also sufficient for
mRNA 3'-end formation in yeast. However, when the
efficiency element is absent or lacks the optimum sequence, low
levels of mRNA having the corresponding poly(A) site are still
observed. It is believed that a portion of the mRNA molecules
terminate at this site, whereas the bulk of the mRNA bypasses the
site but can terminate downstream if a stronger signal is present. We
used the term "preferred sites" to denote these discrete sites of
mRNA 3' ends in the cyc1-512 mutant that are used by the
revertants and the normal CYC1+ strain
(65). Apparently, the cbc1-
mutation enhances
3'-end formation at the 630- and 850-nt preferred sites, thereby acting as a suppressor of cyc1-512 without affecting the half-lives
of these short mRNAs.
Numerous trans-acting factors have been shown to play roles
in 3'-end formation in yeast, including approximately 20 gene products that have direct roles in the processing of
pre-mRNAs in vitro (36). In particular, Hrp1p
appears to stabilize the assembly of the cleavage complex at an
authentic poly(A) site (47). Furthermore, Hrp1p directly
interacts with the UAUAUA efficiency element (10, 38,
80). Our results with enhanced 3'-end formation at the 630- and 850-nt preferred sites in the cyc1-512 mRNAs lacking
the efficiency elements suggest that CBC plays a role in the fidelity
of this interaction and that promiscuous 3'-end formation can occur
with mRNAs lacking the nuclear cap binding proteins.
Thus, studies with the upf1-
, cbc1-
, and
cbc2-
mutants led to the following major conclusions:
transcripts with extended 3' untranslated regions could be recognized
as substrates for NMD, even though the mRNAs lack a premature
termination codon in the ORF; Cbc1p and Cbc2p are components of a novel
mRNA decay pathway, which presumably acts in the nucleus; and Cbc1p
and Cbc2p are required for high-fidelity 3'-end formation at certain
sites. It is reasonable to suggest that either the upf1-
or cbc1-
mutations preferentially affect the levels of
certain normal mRNAs. For example, cbc1-
could cause
the overproduction of certain normal genes that are usually subjected
to degradation by a Cbc1p-dependent pathway. Abnormal mRNA levels
could account for diversed and unrelated phenotypes, such as inhibition
of growth by glucose-repressing conditions (see above) (77,
78) and increase in the negative superhelicity of plasmids
(78).
 |
ACKNOWLEDGMENTS |
We thank Michael R. Culbertson (University of Wisconsin) for the
UPF1 disrupter and other UPF1 plasmids; Charles
N. Cole (Dartmouth Medical School) for the rat7-1 and
RAT7+ strains; V. Raju (Department of
Cardiology, University of Rochester) for advice on Northern blot
analysis; Edward Pagani (Pfizer Inc., Groton, Conn.) for a generous
gift of thiolutin; Jay R. Greenberg (Department of Biochemistry and
Biophysics, University of Rochester), Ding-Fang Yun (Cadus
Pharmaceutical Corp., Tarrytown, N.Y.), Alan Sachs (University of
California, Berkeley), Roy Parker (University of Arizona), Scott Butler
(University of Rochester), Stuart Peltz (University of Medicine and
Dentistry of New Jersey), and Allan Jacobson (University of
Massachusetts Medical School) for helpful discussions; and Satarupa Das
for assistance in the preparation of the cbc2-
disruptant.
This investigation was supported by NIH research grant GM12702 (to
F.S.) and by an FCAR Canadian Postdoctoral Fellowship (to P.C.).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Biophysics, Box 712, University of Rochester Medical School, Rochester, NY 14642. Phone: (716) 275-6647. Fax: (716) 275-6007. E-mail: Fred_Sherman{at}urmc.rochester.edu.
Present address: Division of Biology, California Institute of
Technology, Pasadena, CA 91125.
 |
REFERENCES |
| 1.
|
Alani, E.,
L. Cao, and N. Kleckner.
1987.
Method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains.
Genetics
116:541-545[Abstract/Free Full Text].
|
| 2.
|
Amberg, D. C.,
L. A. Goldstein, and C. N. Cole.
1992.
Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.
Genes Dev.
6:1173-1189[Abstract/Free Full Text].
|
| 3.
|
Atkin, A. L.,
L. R. Schenkman,
M. Eastham,
J. N. Dahlseid,
M. J. Lelivelt, and M. R. Culbertson.
1997.
Relationship between yeast polyribosomes and Upf proteins required for nonsense mRNA decay.
J. Biol. Chem.
272:22163-22172[Abstract/Free Full Text].
|
| 4.
|
Atkin, A. L.,
N. Altamura,
P. Leeds, and M. R. Culbertson.
1995.
The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.
Mol. Biol. Cell.
6:611-625[Abstract].
|
| 5.
|
Belgrader, P.,
J. Cheng, and L. E. Maquat.
1993.
Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA.
Proc. Natl. Acad. Sci. USA
90:482-486[Abstract/Free Full Text].
|
| 6.
|
Briggs, M. W.,
K. T. Burkard, and J. S. Butler.
1998.
Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8S rRNA 3' end formation.
J. Biol. Chem.
273:13255-13263[Abstract/Free Full Text].
|
| 7.
|
Burkard, K. T., and J. S. Butler.
2000.
A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with poly(A)-polymerase and the hnRNA protein Npl3p.
Mol. Cell. Biol.
20:604-616[Abstract/Free Full Text].
|
| 8.
|
Cali, B. M.,
S. L. Kuchma,
J. Latham, and P. Anderson.
1999.
smg-7 is required for mRNA surveillance in C. elegans.
Genetics
151:605-616[Abstract/Free Full Text].
|
| 9.
|
Caponigro, G., and R. Parker.
1996.
Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae.
Microbiol. Rev.
60:233-249[Free Full Text].
|
| 10.
|
Chen, S., and L. E. Hyman.
1998.
A specific RNA-protein interaction at yeast polyadenylation efficiency elements.
Nucleic Acids Res.
26:4965-4974[Abstract/Free Full Text].
|
| 11.
|
Colot, H. V.,
F. Stutz, and M. Rosbash.
1996.
The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.
Genes Dev.
10:1699-1708[Abstract/Free Full Text].
|
| 12.
|
Culbertson, M. R.
1999.
RNA surveillance, unforeseen consequences for gene expression, inherited genetic disorders and cancer.
Trends Genet.
15:74-80[CrossRef][Medline].
|
| 13.
|
Del Priore, V.,
C. Heath,
C. Snay,
A. MacMillan,
L. Gorsch,
S. Dagher, and C. Cole.
1997.
A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae.
J. Cell Sci.
110:2987-2999[Abstract].
|
| 14.
|
Dumont, M. E.,
A. J. Mathews,
B. T. Nall,
D. C. Eustice, and F. Sherman.
1990.
Differential stability of two apo-isocytochrome c in the yeast Saccharomyces cerevisiae.
J. Biol. Chem.
256:2733-2739.
|
| 15.
|
Flaherty, S. M.,
P. C. E. Fortes,
I. W. Mattaj, and G. M. Gilmartin.
1997.
Participation of the nuclear cap binding complex in pre-mRNA 3' processing.
Proc. Natl. Acad. Sci. USA
94:11893-11898[Abstract/Free Full Text].
|
| 16.
|
Görlich, D.,
R. Kraft,
S. Kostka,
F. Vogel,
E. Hartmann,
R. A. Laskey,
I. W. Mattaj, and E. Izaurralde.
1996.
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