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
Beelman, C. A., A. Stevens, G. Caponigro, T. E. LaGrandeur, L.
Hatfield et al., 1996 An essential component of the decapping
enzyme required for normal rates of mRNA turnover. Nature
Benard, L., K. Carroll, R. C. Valle, D. C. Masison and R. B. Wickner,
1999 The ski7 antiviral protein is an EF1-alpha homolog that
blocks expression of non-Poly(A) mRNA in Saccharomyces cere-
visiae. J. Virol. 73: 2893–2900.
Braun, M. A., P. J. Costa, E. M. Crisucci and K. M. Arndt,
2007Identification of Rkr1, a nuclear RING domain protein
with functional connections to chromatin modification in Sac-
charomyces cerevisiae. Mol. Cell. Biol. 27: 2800–2811.
Decker, C. J., and R. Parker, 1993
ble and unstable mRNAs in yeast: evidence for a requirement for
deadenylation. Genes Dev. 7: 1632–1643.
Dunckley, T., and R. Parker, 1999
for mRNA decapping in Saccharomyces cerevisiae and contains
a functional MutT motif. EMBO J. 18: 5411–5422.
Dunckley, T., and R. Parker, 2001
Methods Enzymol. 342: 226–233.
Emori, Y., T. Tsukahara, H. Kawasaki, S. Ishiura, H. Sugita et al.,
1991Molecular cloning and functional analysis of three subu-
nits of yeast proteasome. Mol. Cell. Biol. 11: 344–353.
Flanagan, P. M., R. J. Kelleher, III, M. H. Sayre, H. Tschochner
and R. D. Kornberg, 1991 A mediator required for activation
of RNA polymeraseII transcription in vitro. Nature 350: 436–438.
Frischmeyer, P. A., and H. C. Dietz, 1999
mRNA decay in health and disease. Hum. Mol. Genet. 8:
Frischmeyer, P. A., A. van Hoof, K. O’Donnell, A. L. Guerrerio, R.
Parker et al., 2002 An mRNA surveillance mechanism that
eliminates transcripts lacking termination codons. Science 295:
Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles et al.,
2002 Functional profiling of the Saccharomyces cerevisiae ge-
nome. Nature 418: 387–391.
Gietz, R. D., and R. A. Woods, 2002
ium acetate/single-stranded carrier DNA/polyethylene glycol
method. Methods Enzymol. 350: 87–96.
Goldstein, A. L., and J. H. McCusker, 1999
drug resistance cassettes for gene disruption in Saccharomyces
cerevisiae. Yeast 15: 1541–1553.
Hallier, M., N. Ivanova, A. Rametti, M. Pavlov, M. Ehrenberg
et al., 2004Pre-binding of small protein B to a stalled ribosome
triggers trans-translation. J. Biol. Chem. 279: 25978–25985.
He, F., S. W. Peltz, J. L. Donahue, M. Rosbash and A. Jacobson,
1993 Stabilization and ribosome association of unspliced pre-
mRNAs in a yeast upf1- mutant. Proc. Natl. Acad. Sci. USA 90:
Hsu, C. L., and A. Stevens, 1993
nuclease 1 contain mRNA species that are poly(A) deficient and
partially lack the 59 cap structure. Mol. Cell. Biol. 13: 4826–4835.
Hughes, T. R., C. J. Roberts, H. Dai, A. R. Jones, M. R. Meyer et al.,
2000Widespread aneuploidy revealed by DNA microarray ex-
pression profiling. Nat. Genet. 25: 333–337.
Huh, W. K., J. V. Falvo, L. C. Gerke, A. S. Carroll, R. W. Howson
et al., 2003 Global analysis of protein localization in budding
yeast. Nature 425: 686–691.
Inada, T., and H. Aiba, 2005 Translation of aberrant mRNAs lack-
ing a termination codon or with a shortened 39-UTR is repressed
after initiation in yeast. EMBO J. 24: 1584–1595.
Ito-Harashima, S., K. Kuroha, T. Tatematsu and T. Inada,
2007 Tanslation of the poly(A) tail plays crucial roles in non-
stop mRNA surveillance via translation repression and pro-
tein destabilization by proteasome in yeast. Genes Dev. 21:
Jacobs Anderson, J. S., and R. Parker, 1998
tion of yeast mRNAs is a general mechanism for mRNA turnover
that requires the SKI2 DEVH box protein and 39 to 59 exonu-
cleases of the exosome complex. EMBO J. 17: 1497–1506.
Johnson, A. W., and R. D. Kolodner, 1995
sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces
cerevisiae is independent of killer virus and suggests a general
role for these genes in translation control. Mol. Cell. Biol. 15:
A turnover pathway for both sta-
The DCP2 protein is required
Yeast mRNA decapping enzyme.
Transformation of yeast by lith-
Three new dominant
Yeast cells lacking 59/39 exoribo-
The 39 to 59 degrada-
Synthetic lethality of
Karzai, A. W., M. M. Susskind and R. T. Sauer, 1999
unique RNA-binding protein essential for the peptide-tagging
activity of SsrA (tmRNA). EMBO J. 18: 3793–3799.
Keiler, K. C., P. R. Waller and R. T. Sauer, 1996
tagging system in degradation of proteins synthesized from dam-
aged messenger RNA. Science 271: 990–993.
Kelleher, R. J., III, P. M. Flanagan and R. D. Kornberg, 1990
novel mediator between activator proteins and the RNA polymer-
ase II transcription apparatus. Cell 61: 1209–1215.
Larimer, F. W., C. L. Hsu, M. K. Maupin and A. Stevens,
1992Characterization of the XRN1 gene encoding a 59/39
exoribonuclease: sequence data and analysis of disparate protein
and mRNA levels of gene-disrupted yeast cells. Gene 120: 51–57.
Liu, H. Y., Y. C. Chiang, J. Pan, J. Chen, C. Salvadore et al.,
2001Characterization of CAF4 and CAF16 reveals a functional
connection between the CCR4-NOT complex and a subset of
SRB proteins of the RNA polymerase II holoenzyme. J. Biol.
Chem. 276: 7541–7548.
Lydall, D., and T. Weinert, 1997
charomyces cerevisiae: further evidence for roles in DNA replica-
tion and/or repair. Mol. Gen. Genet. 256: 638–651.
Macbeth, M. R., H. L. Schubert, A. P. Vandemark, A. T. Lingam,
C. P. Hill et al., 2005Inositol hexakisphosphate is bound in
the ADAR2 core and required for RNA editing. Science 309:
Mayer, S. A., and C. L. Dieckmann, 1991
formation is regulated during the induction of mitochondrial
function. Mol. Cell. Biol. 11: 813–821.
Muhlrad, D., and R. Parker, 1992
deadenylation of the yeast MFA2 transcript. Genes Dev. 6: 2100–
Muhlrad, D., and R. Parker, 1994
nation triggers mRNA decapping. Nature 370: 578–581.
Muhlrad, D., and R. Parker, 1999
‘‘nonsense containing’’ leads to both inhibition of mRNA trans-
lation and mRNA degradation: implications for the control of
mRNA decapping. Mol. Biol. Cell 10: 3971–3978.
Muhlrad, D., C. J. Decker and R. Parker, 1995
nisms of the stable yeast PGK1 mRNA. Mol. Cell. Biol. 15: 2145–
Pulak, R., and P. Anderson, 1993
rhabditis elegans smg genes. Genes Dev. 7: 1885–1897.
Ramos, P. C., J. Hockendorff, E. S. Johnson, A. Varshavsky and R.
J. Dohmen, 1998 Ump1p is required for proper maturation of
the 20S proteasome and becomes its substrate upon completion
of the assembly. Cell 92: 489–499.
Rockmill, B., E. J. Lambie and G. S. Roeder, 1991
ment. Methods Enzymol. 194: 146–149.
Seminara, S. B., S. Messager, E. E. Chatzidaki, R. R. Thresher, J. S.
Acierno, Jr. et al., 2003 The GPR54 gene as a regulator of
puberty. N. Engl. J. Med. 349: 1614–1627.
Shyu, A. B., J. G. Belasco and M. E. Greenberg, 1991
destabilizing elements in the c-fos message trigger deadenylation
as a first step in rapid mRNA decay. Genes Dev. 5: 221–231.
Sikorski, R. S., and P. Hieter, 1989
yeast host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122: 19–27.
Sparks, K. A., and C. L. Dieckmann, 1998
choice of several yeast mRNAs. Nucleic Acids Res. 26: 4676–4687.
Steiger, M., A. Carr-Schmid, D. C. Schwartz, M. Kiledjian and R.
Parker, 2003Analysis of recombinant yeast decapping enzyme.
RNA 9: 231–237.
Taniguchi, A., M. Hakoda, H. Yamanaka, C. Terai, K. Hikiji et al.,
1998 A germline mutation abolishing the original stop codon
of the human adenine phosphoribosyltransferase (APRT) gene
leads to complete loss of the enzyme protein. Hum. Genet.
and TATA-binding protein in yeast. Cell 73: 1361–1375.
Tong, A. H., M. Evangelista, A. B. Parsons, H. Xu, G. D. Bader
et al., 2001Systematic genetic analysis with ordered arrays of
yeast deletion mutants. Science 294: 2364–2368.
Tucker, M., M. A. Valencia-Sanchez, R. R. Staples, J. Chen, C. L.
Denis et al., 2001 The transcription factor associated Ccr4
Role of a peptide
G2/M checkpoint genes of Sac-
Yeast CBP1 mRNA 39 end
Mutations affecting stability and
Premature translational termi-
Recognition of yeast mRNAs as
mRNA surveillance by the Caeno-
A system of shuttle vectors and
Novel Aspects of Nonstop mRNA Metabolism783
and Caf1 proteins are components of the major cytoplasmic
mRNA deadenylase in Saccharomyces cerevisiae. Cell 104:
Ueda, K., Y. Yamamoto, K. Ogawa, T. Abo, H. Inokuchi et al.,
2002Bacterial SsrA system plays a role in coping with unwanted
translational readthrough caused by suppressor tRNAs. Genes
Cells 7: 509–519.
van Hoof, A., P. A. Frischmeyer, H. C. Dietz and R. Parker,
mRNAs lacking a termination codon. Science 295: 2262–2264.
Verma, R., S. Chen, R. Feldman, D. Schieltz, J. Yates et al.,
proteasome-interacting proteins by mass spectrometric analysis
of affinity-purified proteasomes. Mol. Biol. Cell 11: 3425–3439.
York, J. D., A. R. Odom, R. Murphy, E. B. Ives and S. R. Wente,
1999A phospholipase C-dependent inositol polyphosphate
kinase pathway required for efficient messenger RNA export.
Science 285: 96–100.
Zaret, K. S., and F. Sherman, 1984
yeast CYC1 mRNA affect transcript stability and translational
efficiency. J. Mol. Biol. 177: 107–135.
Mutationally altered 39 ends of
Communicating editor: S. Gottesman
784 M. A. Wilson, S. Meaux and A. van Hoof