Copyright ? 2007 by the Genetics Society of America
A Genomic Screen in Yeast Reveals Novel Aspects
of Nonstop mRNA Metabolism
Marenda A. Wilson, Stacie Meaux and Ambro van Hoof1
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030
Manuscript received March 12, 2007
Accepted for publication July 26, 2007
Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-
frame stop codons. The cytoplasmic exosome, a complex of 39–59 exoribonucleases involved in RNA
degradation and processing events, degrades nonstop transcripts. To further understand how nonstop
mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are
reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three
mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the
mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain
the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of
nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,
6-pentakisphosphate as an inhibitor of nonstop mRNA decay.
sion. In many instances, changes in mRNA decay rates
modulate gene expression in response to a variety of
signals. Short-lived mRNAs also allow rapid adjustments
to steady-stateRNAlevels after up-or downregulation of
as a model system have identified two general pathways
in most, if not all, eukaryotes. Normally, the major
deadenylase, Ccr4p, gradually removes the poly(A) tail
and initiates mRNA degradation (Shyu et al. 1991;
Muhlrad and Parker 1992; Tucker et al. 2001). This
triggers two deadenylation-dependent decay pathways.
by Dcp1p and Dcp2p (Decker and Parker 1993; Hsu
and Stevens 1993; Muhlrad et al. 1995; Beelman et al.
1996; Dunckley and Parker 1999; Steiger et al. 2003).
1992; Hsu and Stevens 1993; Muhlrad and Parker
1994). In the second pathway, the transcript body is
degraded from the 39-end by a 39–59 exoribonuclease
complex: the exosome (Muhlrad et al. 1995; Jacobs
Anderson and Parker 1998). Although all mRNAs
appear to be degraded by these two pathways, the rate
ESSENGER RNA turnoverisanimportantprocess
that many organisms use to control gene expres-
at which individual steps occur can vary widely depend-
ing on the mRNA. However, it is currently not known
which mechanisms target this basal degradation ma-
chinery preferentially to some mRNAs.
In addition to affecting the expression of normal cel-
control mechanism to maintain the overall fidelity of
gene expression. Eukaryotes have evolved intricate
mechanisms for gene expression. These intricacies
introduce not only potential points of gene regulation,
While many mechanisms exist to ensure high fidelity of
gene expression, errors can occur that lead to aberrant
mRNAs. Hence, specialized mRNA turnover pathways,
termed mRNA surveillance, degrade these aberrant
mRNAs. mRNA surveillance prevents accumulation of
aberrant, dominant-negative, ortruncated proteinsthat
Interestingly, the same enzymes degrade normal and
aberrant transcripts. Transcripts containing premature
stop codons, retained introns, or extended 39-UTRs are
all targets for the nonsense-mediated decay pathway
(Zaret and Sherman 1984; He et al. 1993; Muhlrad
and Parker 1994, 1999). Rapid degradation of non-
sense transcripts bypasses deadenylation and instead
triggers rapiddecapping(Muhlrad and Parker1994).
Similarly, the exosome, independently of prior dead-
enylation, degrades transcripts that lack all in-frame ter-
mination codons, i.e., nonstop transcripts (Frischmeyer
et al. 2002; van Hoof et al. 2002). Thus, understanding
the molecular mechanisms that are responsible for the
1Corresponding author: University of Texas Health Science Center, 6431
Fannin, MSB 1.212, Houston, TX 77030.
Genetics 177: 773–784 (October 2007)
rapid decay of aberrant transcripts may provide insight
Nonstop mRNAs arise in various ways. One source
is premature polyadenylation due to inaccurate 39-end
processing events or cryptic polyadenylation signals
within the coding region of the transcript (Mayer
and Dieckmann 1991; Sparks and Dieckmann 1998;
Frischmeyer et al. 2002). Mutations or errors in tran-
scription that cause a change in the normal stop codon
It is estimated that ?30% of all human disease alleles
1999). While alleles encoding nonstop transcripts have
not been studied in similar detail, generation of a
of the stop codon in the human adenine phosphoribo-
urolithiasis (Taniguchiet al. 1998). Similarly, mutation
in the normal stop codon of a G-protein-coupled re-
ceptor gene that regulates puberty (GPR54) causes
hypogonadotrophic hypogonadism and sterility in af-
fected individuals (Seminara et al. 2003). In both cases,
the nonstop mutation leads to reduced levels of the
nonstop mRNA and the encoded protein. Importantly,
in hypogonadotrophic hypogonadism, overexpression
of the nonstop GPR54 transcript can produce a func-
tional protein. This observation suggests that partial
inhibition of the nonstop mRNA decay machinery in
these patients may prove to be beneficial.
In the current model for nonstop mRNA decay, the
ribosome continues translation to the end of the
poly(A) tail of nonstop transcripts (van Hoof et al.
2002). Upon reaching the end of the transcript, the
ribosome stalls. This stalled ribosome is thought to be
recognized by the C-terminal domain of the Ski7p. This
hypothesis is based on the similarity of this domain to
eEF1A and eRF3, which are known to interact with the
ribosome during translation elongation and termina-
tion, respectively (Benard et al. 1999; van Hoof et al.
2002). Consistent with this hypothesis, this C-terminal
domain is necessary for nonstop mRNA decay, but not
for other exosome functions (van Hoof et al. 2002). In
contrast, the N-terminal domain of Ski7p physically
interacts not only with the exosome, but also with a
complex of Ski2p, Ski3p, and Ski8p (Araki et al. 2001).
nonstop mRNA–ribosome complex, resulting in degra-
dation of the nonstop mRNA (van Hoof et al. 2002).
Recently, it has been shown that proteins encoded by
several nonstop reporters fail to accumulate, which
cannot be fully explained by nonstop mRNA degrada-
tion(Inada andAiba2005; Ito-Harashima etal.2007).
This suggeststhat additionalmechanisms existbywhich
nonstop mRNAs are downregulated. To address the
possibility that there may be additional factors required
to identify factors required for any other aspects of
nonstop mRNA metabolism, we used a genomic screen
in S. cerevisiae. Here, we show that, in addition to the
Ski7p, Ski2p, Ski3p, Ski8p, and the exosome, there are
indeed additional trans-acting factors that are required
for the efficient recognition or degradation of nonstop
mRNA transcripts. Additionally, we provide evidence
that the proteasome degrades the translated nonstop
protein, which may explain why the nonstop protein
fails to accumulate.
MATERIALS AND METHODS
Plasmids: Plasmid pAV188 has been described previously
(van Hoof et al. 2002). It contains a his3-nonstop reporter
gene, a URA3 selectable marker, and a centromere. Plasmid
pAV240 is identical to pAV188 except that it contains a LEU2
selectable marker gene instead of URA3. pAV240 was created
by digesting pAV188 with BamHI and SacI to isolate the his3-
nonstop reporter. The digested his3-nonstop reporter was
cloned into the BamHI and SacI site of pRS415 (Sikorski and
Hieter 1989). Plasmid pAV184 contains a Protein A-nonstop
reporter gene under control of the GAL1 promoter and with a
PGK1 39-UTR. It was created by PCR amplifying the PGK1–
nonstop 39-UTR with oligonucleotides oRP1073 (cgacgggatc
cggtaaggaattgccaggtgtt) and oRP1074 (ggccagtgccaagctttaacg)
from the PGK1-nonstop plasmid described by Frischmeyer
et al. (2002). The resulting PCR product was digested with
BamHI and HindIII and cloned into the BamHI and HindIII
sites of pAV182. Plasmid pAV185 contains a Protein A (with a
stop codon) reporter gene under the control of the GAL1
promoter with a PGK1 39-UTR. It was created by the same
method used to create plasmid pAV184 with the exception
that wild-type PGK1 39-UTR was used for PCR amplification.
The pRS416 plasmid has been previously described (Sikorski
andHieter 1989). pAV182 wasobtained fromRhettMichelson
and Ted Weinert (University of Arizona). pAV182 is a deriva-
tive of pMOV with two Z domains of Protein A under the con-
trol of the GAL1 promoter (Lydall and Weinert 1997).
Transformation and mutant screen: To identify additional
trans-acting factors in nonstop mRNA metabolism, we ob-
transformed each individual strain with pAV188. Transforma-
tion was carried out by a modified version of a previously
described protocol (Gietz and Woods 2002). Briefly, cells
were grown on a YPD plate and transferred to a 96-well plate
containing 10 mg of carrier DNA and 0.5 mg of pAV188 in a
total volume of 10 ml. Next, 150 ml of PLATE solution was
added (40% PEG 3350, 0.1 m lithium acetate, 10 mm TRIS–
HCl, pH 8.0, 1 mm EDTA) and the plate was vortexed and
incubatedatroomtemperature (1 hrtoovernight). Cellswere
heat-shocked for 15 min at 42?, pelleted, resuspended in 10 ml
water, spotted on SC–URA, and incubated for 5 days at 30? to
select for transformants. Transformants were then replica
plated onto SC–HIS and incubated for 3 days at 30? to identify
genes that suppress the his3-nonstop phenotype. Most strains
yielded URA1 transformants on the first attempt. For those
strains where the first transformation failed, a second attempt
to transform was made. Overall, 99% of strains were success-
Rescreen by serial dilution: Strainsthatsuppressedthehis3-
nonstop phenotype were rescreened by individually retrans-
forming these strains with the his3-nonstop reporter using a
standard yeast transformation protocol. Single colonies were
774M. A. Wilson, S. Meaux and A. van Hoof
picked and restreaked onto SC–URA to be used for serial
Serial dilutions were done by growing liquid cultures of
transformants in SC–URA overnight at 30?. The following day,
cells were diluted in SC–URA to a starting OD of 0.2. Cultures
were grown until they reached an OD of 0.8. Cells were serially
diluted in 96-well plates by a factor of 5 and spotted onto SC–
HIS. These plates were then incubated for 3 days at 30? to
qualitatively assay growth relative to wild-type and ski7D
mutants. These experiments were repeated in triplicate.
Confirmation that the His+ phenotype is linked to the
deletion: To ensure that the suppression of his3-nonstop was
indeed caused by the annotated deletion, we PCR amplified
the disrupted gene from the knockout strain, using primers
500 nt on either side of the open reading frame (ORF)
products were used to knock out the genes in BY4741.
Although similar analysis on .30 strains indicated that the
right gene had indeed been deleted, we identified two strains
that were mislabeled in the collection obtained from Open
Biosystems. The two knockouts that were mislabeled were
identified by PCR amplifying and sequencing of the ‘‘molec-
ular barcodes’’ included in the knockout cassettes. We also
identified three cases in which the his3-nonstop suppression
was not recreated, presumably because the phenotype of the
knockout strain was caused by an unlinked mutation.
Stability of Protein A-nonstop mRNA: To determine the
half-life of the Protein A-nonstop mRNA reporter, each strain
was transformed with pAV184. Transformants were grown
overnight in 20 ml of SC–URA12% galactose to induce ex-
pression of the Protein A-nonstop reporter. The following
day, strains were diluted in 50 ml of SC–URA12% galactose to
a starting OD of 0.2 and grown to a final OD of 0.8. Cells were
A 2-ml sample from each strain was taken and pelleted and
stored immediately on dry ice. The remaining liquid culture
was incubated on a shaker at 30?. A total of 2 ml of 40% dex-
trose was addedtoeach strainand2-ml samples were taken(as
above) at the 1-, 2-, 3-, 4-, 6-, 8-, 10-, 15-, 30-, and 60-min time
points.Next,RNAwasisolated fromeachsampleand Northern
blot analysis was performed.
Stability of Protein A-nonstop protein: To determine the
half-life of the Protein A-nonstop protein, wild-type (yAV670)
and proteasome-defective (pre9D, yAV720) yeast strains trans-
formed with pAV184 were grown to midlog phase in media
containing galactose. At this point, transcription and trans-
lation were terminated by replacement with media containing
4% glucose and 100 mg/ml cycloheximide, respectively.
Aliquots were taken at the times indicated and total protein
was isolated. Western blot analysis was performed with anti-
bodies specific for Protein A (Sigma, St. Louis) and Pgk1p
(Molecular Probes, Eugene, OR). Signals were detected by
chemiluminescence (Amersham, Piscataway, NJ), scanned
using a Phosphoimager (Amersham), and quantitated using
Creation of the ski7D pre9D and ipk1D ipk2D double
HygMX4) were created as previously described (Goldstein
and McCusker 1999). yAV720 (pre9DTKanMX4) was mated
with yAV987, and yAV1054 (ipk2DTKanMX4) was mated with
yAV1052. Haploid progeny spores were obtained by the hy-
drophobic spore isolation method essentially as described
(Rockmill et al. 1991) and plated on YPD. Double-mutant
strains were identified by replica plating on YPD1geneticin
Creation of dcp2-7tsdouble mutants: Although DCP2 is
this annotation is incorrect. This conclusion is based on our
unpublished observation that the heterozygous diploid dcp2D
strain 22958 (Open Biosystems) gives two wild-type and two
very slow-growing dcp2D spores per tetrad. To introduce the
dcp2-7tstemperature-sensitive allele into the same genetic
background as the knockout collection, strain 22958 was
transformed with pRP989 (Dunckley and Parker 2001).
The resulting URA1geneticin-sensitive strain was sporulated
to yield yAV747 (Table 1). Strain yAV747 was crossed with
Y3656 (Tonget al. 2001) to give yAV760.
To create yeast deletions strains that also contained a
temperature-sensitive mutation in the decapping machinery,
we mated the yeast deletion strains with strain yAV760.
Haploid progeny spores were obtained by the hydrophobic
spore isolation method essentially as described by Rockmill
et al. (1991) and plated on CSM –Arg –Ura –His plus
canavanine at 23? to select for MATa dcp2-7tsprogeny. MATa
dcp2-7tsprogeny were then replica plated to YPD1geneticin to
identify progeny that also contained the deletion of interest.
To determine whether the strains that we identified in our
deleted for an ORF of interest were grown in YPD overnight at
23?. The following day, cells were diluted to an OD of 0.2 and
grown to an OD of 0.8. Cells were then serially diluted in 96-
well plates by a factor of 5 and spotted onto YPD media plates
and grown for 3 days at 23?, 30?, and 37?. These experiments
were done in triplicate.
Other methods: Western and Northern blotting were done
according to standard methods. Western blots were probed
with an antibody against Protein A (Sigma) or the loading
control Pgk1p (molecular probes). Northern blots were
MATa, his3D1, leu2D0, ura3D0, met15D0
MATa, his3D1, leu2D0, ura3D0, lys2D0
MATa, his3D1, leu2D0, ura3D0, met15D0, lys2D0, dcp2-7TURA3, can1DTMFA1-HIS3TMFa1-LEU2
MATa, his3D1, leu2D0, ura3D0, met15D0, lys2D0, dcp2-7TURA3
MATa, his3D1, leu2D0, ura3D0, lys2D0, ski7DTHYG
MATa, his3D1, leu2D0, ura3D0, lys2D0, ski7DTHYG, pre9TNEO
MATa, his3D1, leu2D0, ura3D0, met15D0, ipk1DTHYG
MATa, his3D1, leu2D0, ura3D0, met15D0, ipk1DTHYG, ipk2TNEO
MATa, his3D1, leu2D0, ura3D0, lys2D0, ipk2TNEO
Novel Aspects of Nonstop mRNA Metabolism775
probed for Protein A using oligo oAV72 (tctactttcggcgcctgag
catcattt) and for the 7S RNA subunit of the signal recognition
particle using oRP100 (gtctagccgcgaggaagg).
A genomic screen identifies mutants that suppress a
his3-nonstop mutation: Nonstop mRNA decay is not an
essential process in yeast (van Hoof et al. 2002). In
addition, mutations that inactivate nonstop mRNA
decay partially suppress a his3-nonstop allele. Specifi-
cally, van Hoof et al. (2002) showed that a wild-type
histidine. However, a ski7D mutant containing the same
his3-nonstop allele is no longer auxotrophic for histi-
dine, presumably because the his3-nonstop mRNA is
stabilized and produces enough His3p for histidine
biosynthesis (van Hoof et al. 2002). With this knowl-
edge, and to expand our understanding of nonstop
mRNA decay, a genetic screen utilizing a deletion
collection of almost 5000 nonessential open reading
frames in S. cerevisiae was used to identify additional
genes involved in the nonstop mRNA decay pathway.
HIS3 genes and a complete deletion in a nonessential
openreading frame. Eachstrain from thecollectionwas
individually transformed with a plasmid containing a
selectable URA3 gene and a his3-nonstop reporter.
Transformants were selected by growth in the absence
of uracil and replica plated onto media lacking histi-
dine. Potential genes involved in nonstop mRNA decay
were identified on the basis of the ability of the mutant
to grow on media lacking histidine. To eliminate false
positives, we restreaked each strain on media lacking
uracil and selected single colonies. These strains were
placed in 96-well plates with fivefold serial dilutions and
plated onto medialacking histidineor uracil. The trans-
formation was then repeated using a high-efficiency
transformation protocol to eliminate additional false
positives. This screen yielded a number of mutants that
reproducibly suppressed the his3-nonstop phenotype to
varying extents. Here, we concentrated on the mutants
that increased his3-nonstop expression approximately
as much as a ski7D mutant. In addition to these, we
isolated several mutants that showed small increases in
growth, but grew significantly slower than the ski7D
control (data not shown).
The his3-nonstop suppression phenotype is tightly
linked to the deletion: During creation and mainte-
nance of the deletion collection, some strains may have
been mislabeled or may have accumulated unlinked
mutations (e.g., Hughes et al. 2000). Therefore, to en-
sure that the observed his3-nonstop suppression pheno-
type was indeed caused by the deletion, we recreated
each of the newly identified mutants in the wild-type
strain BY4741. As controls, we included the ski3D, ski7D,
and ski8D mutants. Indeed, this analysis identified two
strains that were mislabeled in the knockout collection
and three strains where the original his3-nonstop sup-
pression phenotype was not tightly linked to the de-
letion. Presumably, these latter three strains from the
collection contain an unlinked mutation that sup-
pressed the his3-nonstop phenotype. However, for 15
nonstop suppression phenotype was indeed linked to
their deletion mutants (Figure 1 and Table 2).
Many his3-nonstop suppressing mutations also in-
crease the abundance of a Protein A-nonstop mRNA:
There are at least two explanations for the suppression
of the his3-nonstop phenotype in the mutants that we
identified. One possibility is that the phenotype is
specific to the HIS3 gene. For example, any mutation
that dramatically increases transcription from the HIS3
promoter couldresultin histidineautotrophy.A second
due to a general defect in the nonstop mRNA degrada-
tion pathway. In this latter case, expression of all
nonstop mRNAs in the cell would be increased in the
mutant. To distinguish between these two possibilities,
we analyzed the effect of the mutations on a second
nonstop reporter. This Protein A-nonstop reporter
shares no sequence homology with the his3-nonstop
Figure 1.—Isolation of deletion mutants of S.
cerevisiae that suppress a his3-nonstop reporter.
Cells containing a his3-nonstop reporter as the
only source of His3p grow slowly on plates lacking
histidine. The yeast deletion collection was trans-
formed with a his3-nonstop reporter. To assay sup-
pression of the his3-nonstop phenotype, each of
the indicated strains was serially diluted and spot-
ted on media lacking histidine.
776M. A. Wilson, S. Meaux and A. van Hoof
gene. Specifically, the his3-nonstop reporter contains
the HIS3 promoter, HIS3 coding region, and HIS3 39-
UTR,while theProteinA-nonstop reportercontainsthe
GAL1promoter,ProteinA-codingregion, and thePGK1
39-UTR. After transforming each one of our deletion
mutants with the Protein A-nonstop reporter, RNA was
isolated and Protein A-nonstop mRNA levels were anal-
yzed relative to wild type and ski7D controls (Figure 2).
This experiment showed that deletion of 11 of the 15
mutants tested also increased the abundance of our
Importantly, these data showed that suppression in
these 11 strains was not specific to the his3-nonstop
reporter, but was most likely due to a general defect in
the nonstop mRNA decay pathway.
Mutations implicating the proteasome increase Pro-
tein A-nonstop protein, but without a corresponding
increase in mRNA levels: The above analysis indicated
that four of the deletion mutant strains increased his3-
nonstop expression but not Protein A-nonstop mRNA
abundance. Intriguingly, three of these four genes are
implicated in proteasome function, which suggests that
the proteasome may normally degrade the his3-nonstop
protein. Pre9p is a proteasome b-type subunit. It is the
only nonessential component of the 20S proteasome
Genes that suppress the nonstop phenotype
Gene Function/putative function
his3-nonstop Steady stateStability
dcp synthetic lethal
Exosome-mediated mRNA degradation
Exosome-mediated mRNA degradation
Exosome-mediated mRNA degradation
Exosome-mediated mRNA degradation
Proteasome-mediated protein degradation
Proteasome-mediated protein degradation
Proteasome-mediated protein degradation
Subunit of the RNA polymerase II mediator;
interacts with mRNA deadenylase
Kinase of the RNA polymerase II mediator;
interacts with mRNA deadenylase
Subunit of the RNA polymerase II mediator
mRNA export, RNA editing, inositol phosphate
Variant of histone H2
Subunit of the nuclear pore complex
His3-nonstop growth is indicated as ?, indicating little or no growth; 11, indicating significant growth; and 111, indicating
growth that was even more pronounced. dcp2-7 synthetic lethality is indicated with a 1 sign and ? sign indicates no synthetic
growth defect. ND, not determined.
Figure 2.—Many of the genes that suppress
the his3-nonstop phenotype also increase mRNA
levels of a second nonstop mRNA reporter.
Strains isolated as suppressors of his3-nonstop
were transformed with a plasmid containing a
Protein A-nonstop reporter. The steady-state level
of Protein A-nonstop mRNA was assayed by
Northern blot. Shown is the average and the
spread of two independent experiments after cor-
recting for loading difference using a probe for
the RNA subunit of the signal recognition parti-
cle (7S RNA). The mRNA level is expressed as a
value relative to that found in an isogenic wild-
Novel Aspects of Nonstop mRNA Metabolism 777
collection (Emori et al. 1991; Giaever et al. 2002).
Ump1p is a short-lived chaperone that is involved in
the maturation of the proteasome (Ramos et al. 1998).
YMR247C encodes a putative ubiquitin-conjugating
enzyme that copurifies with the 19S regulatory subunit
One possible reason that deletions in the PRE9, UMP1,
and YMR247C genes suppressed the his3-nonstop phe-
notype is that the protein encoded by the his3-nonstop
mRNA is normally rapidly degraded by the proteasome.
In this instance, suppression of the his3-nonstop phe-
notype would not be due to increased stability of the
mRNA, but to increased stability of the protein.
To further characterize the effects of the PRE9, UMP1,
and YMR247C genes on protein levels encoded from
nonstop reporter transcripts, we examined the levels of
Protein A-nonstop protein. Interestingly, far more Pro-
the pre9D, ump1D, and ymr247cD mutants compared to
extracts from wild-type cells (Figure 3). This occurred
despite the relatively low nonstop mRNA levels in these
tion most likely suppressed the his3-nonstop phenotype
because they stabilize the His3-nonstop protein.
From the above analysis, it is not entirely clear when
the proteasome degrades the protein encoded by Pro-
tein A-nonstop mRNA. One possibility is that the protea-
some degrades the protein cotranslationally, while a
second possibility is that the encoded protein is de-
graded after synthesis is completed. To distinguish be-
rateof fullysynthesized ProteinAencodedbyanonstop
mRNA in wild-type and pre9 cells. Cells were grown in
the presence of galactose, and then glucose and cyclo-
heximide were added to prevent further synthesis of
Protein A. Analysis of the levels of Protein A at various
times after this treatment showed that decay from the
steady-state pool was relatively rapid in wild-type cells,
4). Therefore, the pre9 deletion affects the decay of
Protein A out of the steady-state pool. We conclude that
Pre9p, and presumably Ump1p and yMR247Cp, affect
the post-translational degradation of the protein en-
coded by the Protein A-nonstop reporter.
If ski7D suppressed the his3-nonstop phenotype by
nonstop mRNA, and pre9D suppressed his3-nonstop
phenotype by inactivating the proteasome and stabiliz-
ing the nonstop protein, a pre9D ski7D double mutant
might exhibit an additive effect on the suppression
phenotype. This was indeed the case since the ski7D
pre9D double mutant grew better in the absence of
histidine than either the ski7D or pre9D single mutants
when all contained the his3-nonstop reporter (Figure
5A). We also noted that the ski7D pre9D double mutant
grew slower in the presence of histidine than either
single mutant. This genetic interaction was consistent
with the hypothesis that SKI7 and PRE9 genes may act in
pathways that are functionally related. In addition,
Northern and Western blot analysis of the ski7D pre9D
double mutant showed that while Protein A-nonstop
mRNA levels are not significantly increased, it accumu-
mutant alone (Figure 5, B and C). We conclude that
ski7D and pre9D suppressed the his3-nonstop phenotype
by independent mechanisms (see discussion).
Many his3-nonstop suppressing mutations decrease
the rate of Protein A-nonstop mRNA decay: While our
screen was designed to isolate mutants that stabilize the
his3-nonstop mRNA, other types of mutants might also
suppress the his3-nonstop phenotype. For example,
mutants that increase transcription from the HIS3 pro-
moter could also result in higher levels of his3-nonstop
Figure 3.—Mutations implicating the proteasome increase
the steady-state protein level of a nonstop reporter, without
increasing the steady-state mRNA level. (Top) Total RNA
was isolated from yeast strains, and Protein A-nonstop mRNA
levels were analyzed by Northern blot. The RNA subunit of
the signal recognition particle (7S RNA) served as a loading
control. (Bottom) Protein was isolated from yeast strains ex-
pressing Protein A-nonstop protein and analyzed by Western
blot. Pgk1p served as a loading control.
Figure 4.—The pre9 deletion increases the stability of the
protein encoded by the Protein A-nonstop reporter. Wild-type
and pre9D strains were transformed with a Protein A-nonstop
reporter under the control of a galactose-inducible promoter.
Expression of the reporter was repressed by the addition of
glucose and cycloheximide. The stability of the Protein A-
nonstop protein was determined by Western blot with anti-
bodies specific to Protein A. Pgk1p served as a loading control.
778 M. A. Wilson, S. Meaux and A. van Hoof
mRNA and thus in increased growth in the absence of
histidine. This latter possibility is especially relevant for
three of the mutants identified in our screen that have
previously been implicated in transcription: ssn2, ssn3,
and med1. These genes have been implicated in both
transcriptional activation and repression of a variety of
genes, suggesting that they might increase his3-nonstop
expression by increasing the strength of the HIS3 pro-
moter (Kelleher et al. 1990; Flanagan et al. 1991;
Thompson et al. 1993). On the other hand, Ssn2p and
Ssn3p interact with the CCR4/NOT complex, which is
the major cytoplasmic deadenylation complex, suggest-
ing that they might have a direct role in mRNA decay
(Liu et al. 2001; Tuckeret al. 2001; Huh et al. 2003).
We directly measured Protein A-nonstop mRNA de-
cay rates to distinguish between increased transcription
and increased mRNA stability in all mutants that did
not implicate proteasome function. In this experiment,
mRNA was isolated at various time points after tran-
scription of the Protein A-nonstop reporter was re-
pressed by the addition of glucose and the half-life of
These measurements were repeated three or four times
and the average is plotted in Figure 6. As expected, the
decay rate of Protein A-nonstop mRNA was about
threefold slower in ski3D, ski7D, and ski8D strains than
in wild-type cells. Interestingly, the eight other mutants
isolated in our genomic screen also increased the
stability of the Protein A-nonstop mRNA, although to
a lesser extend than the ski deletions. The largest
increase in Protein A-nonstop half-life (approximately
twofold) was observed for the deletion of yLR021w, an
uncharacterized open reading frame of unknown func-
tion. These data are consistent with the hypothesis that
the deletions that we identified indeed increased the
half-life of nonstop reporter mRNAs rather than in-
is that these mutants increase transcription from the
nonstop reporter and that this increased transcription
saturates the nonstop mRNA decay machinery, in turn
leading to an increased mRNA stability. To determine
whether or not we could saturate the nonstop mRNA
decay pathway, we introduced the Protein A-nonstop
reporter, which is expressed from the strong GAL1
promoter into a strain that also contains a his3-nonstop
reporter expressed from the HIS3 promoter. As a con-
trol, we used a plasmid that encodes a normal Protein A
mRNA from the GAL1 promoter. The production of
Protein A-nonstop mRNA did not affect the growth
phenotype of the his3-nonstop mutation (Figure 7). We
Figure 5.—The effects of ski7D and pre9D on nonstop gene
expression are additive. (A) Yeast strains containing muta-
tions in the exosome (ski7D), proteasome (pre9D), or the exo-
some and proteasome (ski7D, pre9D) were transformed with a
his3-nonstop reporter, serially diluted, and spotted on media
without histidine to assay for suppression of the his3-nonstop
phenotype. The same strains were transformed with a Protein
A-nonstop reporter and analyzed by Northern blotting (B)
and Western blotting (C).
Figure 6.—Mutants that increase steady-state
levels of the Protein A-nonstop mRNA also in-
strains was transformed with a plasmid directing
hibiting transcription from the GAL1 promoter
with the addition of glucose and isolating RNA
atvarious points after transcription inhibition was
quantitated. Plotted is the average mRNA remain-
mutant and from four independent experiments
for the wild type.
Novel Aspects of Nonstop mRNA Metabolism 779
conclude that the nonstop mRNA decay pathway is not
easily saturated. Therefore, these data indicate that the
increased expression of his3-nonstop and Protein A-
nonstop in mediator mutants is unlikely to result from
the saturation of the nonstop mRNA decay machinery.
suppresses his3-nonstop: One of the deletions that
suppressed the phenotype of his3-nonstop was ipk1D.
The IPK1 gene encodes the enzyme inositol 1,3,4,5,6-
pentakisphosphate 2-kinase that produces inositol
1,3,4,5,6-pentakisphosphate (IP5) (York et al. 1999).
IP6 has known roles in regulating RNA metabolism.
Yeast mutants lacking IP6 accumulate poly(A)1RNAs in
the nucleus and have defects in tRNA modification
(York et al. 1999; Macbeth et al. 2005). To investigate
whether the role of Ipk1p in his3-nonstop suppression
was related to these previously known functions of
Ipk1p, we analyzed other mutants lacking IP6. IP6
production is a four-step metabolic pathway (Figure
8A) that also requires phospholipase C (Plc1p) and
Ipk2p. Strikingly, plc1D and ipk2D mutations were not
identified in our genomic screen and, upon direct test-
ing, had no effect on his3-nonstop expression. In con-
trast, all other known defects of ipk1D mutants are
his3-nonstop expression is not due to a lack of IP6.
A second possible mechanism by which ipk1D affects
his3-nonstop expression is that the mutant accumulates
1999). This hypothesis predicts that Ipk1p can no longer
affect his3-nonstop expression if IP5 accumulation is
prevented by a mutation in IPK2. A third possibility is
that Ipk1p is a bifunctional protein with completely
separate roles in IP6 production and his3-nonstop sup-
pression. Under this hypothesis, we predict that Ipk1p
can still affect his3-nonstop expression in an ipk2 strain.
To distinguish between these latter two possibilities, we
tested suppression of the his3-nonstop phenotype in an
the double mutant does not suppress the his3-nonstop
pression phenotype that we observed in an ipk1D strain
is a result of the accumulation of IP5 and not a second,
unrelated function of Ipk1p. To our knowledge, this
is the first result that implicates IP5 as a regulatory
Some mutations affect nonstop mRNA decay without
affecting other cytoplasmic exosome functions: Muta-
tions such as a ski7D that inactivate the cytoplasmic exo-
some stabilize nonstop mRNAs (van Hoof et al. 2002).
Thus, at least two classes of mutants might be expected
in our screen. One class would have defects in cytoplas-
mic exosome function (e.g., ski7D), while a second class
of nonstop mRNAs (e.g., ski7DC; van Hoof et al. 2002).
Mutations disrupting cytoplasmic exosome function
exhibit synthetic lethality with decapping defects, while
nonstop mRNA recognition mutants would not be ex-
and Parker 1998; van Hoof et al. 2002). We therefore
tested whether the new mutants that we identified were
synthetically lethal with a decapping defect.
Each of the mutants was crossed with a temperature-
sensitive decapping mutant (dcp2-7ts), and double mu-
tants were isolated. At 37?, the dcp2-7tsallele inactivates
the decapping enzyme and thereby inactivates the 59–39
Figure 7.—Overexpression of Protein A-non-
stop does not saturate the nonstop mRNA decay
pathway could easily be saturated, wild-type and
ski7D strains were transformed with plasmids ex-
pressing his3-nonstop from the HIS3 promoter
and either Protein A-nonstop expressed from
the GAL1 promoter or the indicated control plas-
mids. Strains were serially diluted and spotted on
media containing 2% galactose as the sole carbon
source and either lacking or containing histidine.
Figure 8.—IP5 affects the expression of his3-nonstop. (A)
Ipk1p catalyzes the conversion of IP5 to IP6 and acts down-
stream of Plc1p and Ipk2p. (B) ipk2D and ipk1D, ipk2D do
not suppress the his3-nonstop phenotype. ipk1D, ipk2D, and
ipk1D ipk2D deletion strains were transformed with a his3-non-
stop reporter. To assay suppression of the his3-nonstop pheno-
type, each of the indicated strains was serially diluted and
spotted on media lacking or containing histidine.
780M. A. Wilson, S. Meaux and A. van Hoof
are still able to grow at 37?, because the alternative 39–59
decay pathway is intact and sufficient for viability.
However, when the dcp2-7tsallele is combined with a
mutation inactivating the 39–59 decay pathway, both
pathways are nonfunctional at 37?, and therefore such a
strain cannot grow at 37?. Thus, an inability to grow at
the nonpermissive temperature (37?) would suggest
that the gene is required for general exosome-mediated
decay of mRNAs. Strikingly, most of the genes that we
identified in our genetic screen did not have growth
defects at the nonpermissive temperature when com-
observation suggests that these genes are not required
for general cytoplasmic exosome activity.
Although most of the genes tested did not show a
synthetic lethal interaction with dcp2-7ts, three deletions
did significantly reduce growth of the dcp2-7tsstrain and
htz1D, and ylr021wD). As shown in Figure 9, at the
nonpermissive temperature, these cells were either syn-
ing mutation. One explanation for these findingsis that
their function is not limited to nonstop mRNA decay,
but that they have a more general function in cytoplas-
mic exosome function. To more directly determine
whether NUP2, HTZ1, and YLR021W genes function in
exosome-mediateddecayof allmRNAs, we assayed their
effects on stability of the GAL7 and GAL10 mRNAs. We
grew the dcp2-7tsdouble mutants in YEP1galactose to
induce expression of the GAL7 and GAL10 mRNAs. We
then incubated the cells at 37? for 1 hr to inactivate the
decapping enzyme and then added glucose to shut off
transcription of the GAL7 and GAL10 mRNAs. Under
these conditions, cytoplasmic mRNA decay is solely car-
1998). As expected, the GAL7 and GAL10 mRNAs were
stabilized in the ski7D dcp2-7 double mutant, when com-
pared to the dcp2-7 single mutant (Figure 9B). The
nup2D, htz1D, and ylr021wD mutants did not have this
same effect, suggesting that these three genes were not
required for exosome-mediated decay of the GAL7 and
GAL10 mRNAs. However, the ylr021wD mutant ap-
peared to have a minor defect in that the GAL7 mRNA
decayed with biphasic kinetics. Approximately half of
the GAL7 mRNA decayed with normal kinetics (half-life
of 8 min), while the other half was stabilized (half-life
.20 min). While the significance of this biphasic decay
is not understood, we conclude that these three genes
are not required for all functions of the cytoplasmic
exosome. Overall, analysis of dcp2-7tsdouble mutants
suggests that we have identified a number of proteins
that are required for exosome-mediated decay of his3-
nonstop mRNA and Protein A-nonstop mRNA, but not
for all exosome-mediated mRNA decay.
The nonstop mRNA decay pathway identifies and
degrades aberrant transcripts that may encode proteins
Therefore nonstop mRNA decay is a part of the cell’s
mRNA surveillance mechanisms that maintain the over-
Figure 9.—Mutations in Htz1p, Nup2p, and
yLR021wp are synthetic lethal with decapping de-
normal mRNAs. Mutants that block cytoplasmic
exosome function are synthetically lethal with de-
fects in decapping. The majority of the genes iso-
lated in our screen do not show synthetic
lethality (not shown). (A) The htz1D, nup2D, and
ylr021wD genes are synthetically lethal with a tem-
(dcp2-7), consistent with the possibility that they
some function. The indicated strains were serially
diluted and spotted on YPD plates that were then
incubated at 23? or 37?. At 23?, the decapping en-
whether the synthetic lethality shown in A was
caused by a general block of cytoplasmic exosome
sured. The stability of the GAL7 mRNA was ana-
lyzed by growing cells at 25? in media containing
galactose. The decapping enzyme was then inacti-
RNA was used as a loading control. Each time point represents the average of two experiments.
Novel Aspects of Nonstop mRNA Metabolism 781
nonstop mRNAs are removed from eukaryotic cells, we
took a genetic approach in S. cerevisiae. Using the yeast
deletion mutant collection, we identified known and
metabolism. One measure of the completeness of this
screen is that we identified all four nonessential genes
known to be required for nonstop mRNA decay (i.e.,
SKI2, SKI3, SKI7, and SKI8). Importantly, most of the
newly identified genes do not appear to be required for
nonstop mRNA metabolism is more complex than pre-
viously known, involving roles in diverse functions like
proteolysis and phosphoinositide signaling.
The one possible exception to the observation that
the newly identified genes do not affect other exosome
functions may be yLR021w. Unlike most of the newly
identified genes, ylr021wD is synthetically lethal with
decapping defects and has a small effect on the
degradation of GAL7 mRNA. ylr021wD also had the
largest effect on Protein A-nonstop mRNA stability and
thus may encode a regulator of the cytoplasmic exo-
some. The function of yLR021w is completely unchar-
to any protein with a known function.
Our results suggest a role for genes encoding func-
tions for the eukaryotic proteasome in nonstop mRNA
surveillance. The proteasome was implicated three
times in our genetic screen, whichstrongly suggests that
it may be involved in degrading the his3-nonstop pro-
tein. The PRE9 gene encodes a subunit of the 20S
proteasome and is the only nonessential proteasome
gene (Emori et al. 1991; Giaever et al. 2002). The UMP1
assembly (Ramos et al. 1998). The YMR247C ORF
encodes a protein that copurifies with the proteasome
and has very recently been identified as a RING domain
containing ubiquitin-conjugating enzyme (Verma et al.
cause increased abundance of nonstop transcripts but
instead cause increased levels of the nonstop protein
product. Consistent with our conclusion that the his3-
nonstop protein is normally degraded by the protea-
that addition of eight or more lysine residues could
target His3p to proteasome-mediated degradation and
that this proteolysis contributes to the reduced expres-
sion of nonstop reporter genes.
In Eubacteria, the signal that identifies a transcript as
nonstop is thought to arise from the stalled ribosome
(Keiler et al. 1996; Ueda et al. 2002). When this occurs,
an RNP composed of tmRNA and SmpB recognizes the
stalled ribosome and this recognition adds a C-terminal
peptide tag to the protein encoded by the nonstop
mRNA (Keiler et al. 1996; Karzai et al. 1999; Hallier
et al. 2004). The addition of this tag targets the protein
encoded by the nonstop mRNA for rapid proteolysis.
Our identification of three mutants that implicate the
proteasome in the degradation of the his3-nonstop and
Protein A-nonstop proteins suggests the possibility that
eukaryotes may also actively recognize and degrade
proteins encoded by nonstop mRNAs. There are several
ways in which the proteins encoded by the his3-nonstop
and Protein A-nonstop mRNAs might be targeted to the
proteasome. Analogous to the prokaryotic system, the
stalled ribosome at the end of an mRNA could target
the encoded protein for degradation. However, two
lines of evidence do not support this idea. First, Ski7p
most likely plays a central role in recognizing the stalled
ribosome, and analysis of a ski7D pre9D double mutant
clearly shows that even in the absence of the Ski7p, the
his3-nonstop and Protein A-nonstop proteins are still
targeted to the proteasome (Figure 5C). Second, direct
measurement of Protein A-nonstop protein stability
indicates that Pre9p acts post-translationally, rather
than cotranslationally (Figure 4). Future experiments
are required to understand the physiological signifi-
by nonstop mRNAs.
Another unexpected finding in our genetic screen is
the identification of the IPK1 gene, which is involved in
cellular signaling and nuclear transport. The IPK1 gene
encodes the enzyme inositol 1,3,4,5,6-pentakisphos-
phate 2-kinase that produces IP6 from IP5. (York et al.
1999). Analysis of other mutants in the IP6 pathway
implicated IP5 as an inhibitor of his3-nonstop expres-
sion. Most importantly, when IP5 production in the
nonstop suppression was reversed. To our knowledge,
this is the only known role of IP5. In contrast, IP6 has
been implicated in several aspects of RNA metabolism:
ipk1, ipk2, and plc1 mutants accumulate polyadenylated
RNA in the nucleus, and thus IP6 may have a role in
nuclear export of poly(A)1mRNAs (York et al. 1999).
Interestingly, exosome mutants also accumulate poly-
adenylated RNA in the nucleus, suggesting the possibil-
ity that both IP5 and IP6 regulate diverse exosome
functions. In addition, IP6 is an important component
of adenine deaminases that act on mRNA and tRNA
(ADARs and ADATs, respectively), and mutants lacking
IP6 have defects in tRNA modification (Macbeth et al.
2005). Overall, these results suggest that phosphoinosi-
tides might regulate diverse aspects of RNA processing
We thank Roy Parker for his encouragement, helpful discussions,
and suggestions duringthis workand Tom Vida forhis critical reading
of the manuscript. This work was funded by the National Institutes of
toA.V. andby anAmerican Society for Microbiology Robert D.Watkins
Fellowship to M.A.W.
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Communicating editor: S. Gottesman
784 M. A. Wilson, S. Meaux and A. van Hoof