Gene Overexpression as a Tool for Identifying New trans-Acting Factors Involved in Translation Termination in Saccharomyces cerevisiae

Corresponding author: Jean-Pierre Rousset, Institut de Génétique et Microbiologie, bâtiment 400, Université Paris-Sud, 91405 Orsay Cedex, France., rousset{at}igmors.u-psud.fr (E-mail)

Communicating editor: A. NICOLAS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.

TRANSLATION termination in eukaryotes occurs when a stop codon enters the A site of the ribosome and is controlled mainly by a complex composed of eRF1p and eRF3p, respectively, encoded by the SUP45 and SUP35 genes (STANSFIELD et al. 1995 ; ZHOURAVLEVA et al. 1995 ). Polypeptide and hydroxyl-tRNA release activity is provided by eRF1p, while eRF3p is assumed to fulfill the roles of the RF3 and RRF prokaryotic factors (KISSELEV and FROLOVA 1999 ). eRF3p may have other functions, since it interacts with numerous proteins. However, the physiological relevance of these interactions remains unclear, since the various partners exhibit different functions that are not directly interrelated: Upf1p is implicated in the degradation of mRNA carrying a premature stop codon (NMD; LEEDS et al. 1991 ; CZAPLINSKI et al. 1995 ); Mtt1p is an RNA helicase (CZAPLINSKI et al. 2000 ); Atp17p is a subunit of ATP synthase (SHUMOV et al. 2000 ); and Pab1p is the poly(A) binding protein (HOSHINO et al. 1999 ).

Furthermore, mutations in yeast SUP45 or SUP35 genes display various associated phenotypes such as allosuppression (WAKEM and SHERMAN 1990 ), sensitivity to paromomycin, high or low temperature sensitivity, respiratory deficiency (TIKHOMIROVA and INGE-VECHTOMOV 1996 ), and benomyl sensitivity (TIKHOMIROVA and INGE-VECHTOMOV 1996 ). Recently, it has been shown in Saccharomyces cerevisiae that some mutations of SUP35 or SUP45 genes also produce elevated chromosome instability (BORCHSENIUS et al. 2000 ).

To date, genetic screens constructed for the identification of factors involved in translation termination have been performed by the classical suppressor approach: an auxotrophic mutant arising from a stop mutation is used to positively select for revertants. Using these initial mutants, either omnipotent (e.g., release factors) or codon-specific suppressors (e.g., tRNAs), allosuppressors, or antisuppressors can be selected by appropriate screens (see references in TER-AVANESYAN et al. 1994 ). Several readthrough sites have been already described in which naturally occurring stop codon contexts result in an elevated proportion of leaky termination (up to 25%; BONETTI et al. 1995 ; STAHL et al. 1995 ). In viruses, these readthrough events are responsible for the expression of elongated proteins that normally provide the replicase function of the virus (see FARABAUGH et al. 2000 for review). Since readthrough frequency can be very high, it offers the possibility for one-step selection not only for suppressors, but also for antisuppressors. In a number of genetic systems, increased dosage or inappropriate expression of wild-type gene products has been used to decipher complex assembly processes. The overexpression of a wild-type protein participating in a biological process may interfere with that process, either by titration of a diffusible partner or through feedback control.

In this study we present a genetic screen designed to isolate genes whose increased dosage would modify translational readthrough efficiency. The screen was based on a reporter system in which the tobacco mosaic virus (TMV) stop codon leaky context, directing a high readthrough level in yeast, was introduced into the lacZ gene. This construct was integrated into the yeast genome, providing a simple and stringent assay for translation termination efficiency in living cells and allowing one-step screening for both suppressors and antisuppressors.

Twenty-three genomic fragments in which overexpression modified translation termination efficiency were found in this screen, and three were analyzed in more detail. This identified three candidate genes: tRNA^Gln, STU2, and SSO1. Stu2p is a microtubule-binding protein and an essential component of the yeast spindle pole body (WANG and HUFFAKER 1997 ; SEVERIN et al. 2001 ). Sso1p, a protein homologous to syntaxin, is involved in vesicle transport between the Golgi apparatus and the plasma membrane (AALTO et al. 1993 ). Overall, our results indicate a potential connection between protein transport and translation termination in yeast and extend previous observations on a possible link between the cytoskeletal apparatus and the translational process.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strain manipulation:
Strains used in this study were Y349 (MAT lys2201 leu2-3,112 his3200 ura3-52; DANG et al. 1996 ) and its derivative, YONX (MAT lys2201 leu2-3,112 his3200 ura3-52 ASN1::lacZ-UAG). The UAG leaky stop codon was inserted into a vector carrying the lacZ reporter gene flanked by the ASN1 promoter and 530 nucleotides of 3'-untranslated region of the ASN1 gene. To insert the leaky stop codon, three restrictions sites on the lacZ gene were selected, one at the beginning (XbaI site), one in the middle (BssHII site), and one near the 3'-end (SpeI site). Only insertion in the XbaI site gave detectable ß-galactosidase activity (data not shown). This insert is flanked by SpeI/BstEII sites that can be used to isolate a linear fragment. The YONX strain was obtained by homologous recombination at the ASN1 locus, with an ASN1::lacZ-UAG construct (Fig 1). Correct integration was verified by Southern blot using ASN1 and lacZ specific probes and by PCR (data not shown).

View larger version (29K): In this window In a new window Download PPT slide   Figure 1. Construction of the YONX strain. The ASN1 locus was replaced by a construct carrying a lacZ gene requiring a translational readthrough event to be expressed. ASN1 is a constitutively and highly expressed gene. See details in MATERIALS AND METHODS.

Yeast strains were transformed using the lithium acetate method according to ITO et al. 1983 . YONX cells were transformed with a high-expression S. cerevisiae genomic DNA library. Cotransformation of Y349 strain was performed using equal quantities of pAC-TMV reporter plasmid and the overexpression plasmid.

Media and secondary screens:
YNB (0.67% yeast nitrogen base, 2% glucose) was used for standard growth conditions. Each phenotype was tested on plates, using five dilutions (3 x 10⁶, 6 x 10⁵, 1.2 x 10⁵, 2.4 x 10⁴, and 4.8 x 10³ cells) of each transformed strain. Temperature sensitivity was monitored after 24 hr of growth at 37° or 72 hr at 15°. To test the sensitivity to osmotic change, growth was monitored after 36 hr at 30° in the presence of 2 M ethylene glycol. G418 sensitivity was monitored after 72 hr of growth at 30° in the presence of 500 µg/ml of antibiotic.

Plasmids:
The plasmid carrying ASN1-lacZ sequences was kindly provided by Monique Bolotin-Fukuhara (DANG et al. 1996 ). The yeast genomic DNA library was kindly provided by François Lacroute. It was constructed by partial restriction of genomic DNA by Sau3A from the S288c strain and ligation of the fragments into the BamHI site of the pFL44L multicopy plasmid (BONNEAUD et al. 1991 ).

Different sequences corresponding to the three stop codons in different contexts were cloned in the pAC99 dual reporter vector (NAMY et al. 2002 ). Oligonucleotides used to construct the various reporters are given Table 1. The pAC-TMV reporter construct has been previously described (STAHL et al. 1995 ; BIDOU et al. 2000 ).

Subcloning of candidate sequence fragments:
Subcloning was performed by enzymatic restriction of the vector and cloning the insert into the pFL44L vector, in the same orientation as that in the parental vector. The ptRNA plasmid was obtained by cloning a KpnI fragment, containing YDR098c and tRNA-gln, into the KpnI site of the pFL44L. The pVMA11 plasmid was obtained by cloning an Fsp1 fragment, containing VMA11 and YPL233w, into the pFL44L vector. The p468 vector was constructed by PvuII enzymatic restriction and self-ligation of p468. The resulting vector carries the SSO1 and YPL233w open reading frames (ORFs). The coding sequence of the STU2 gene was amplified by PCR from genomic DNA with Platinum Pfx Taq DNA polymerase (Life Technologies), using as primers STUNw (5'-ATGTCAGGAGAAGAAGAAGT-3') and STUCc (5'-TTACGTCCTGGTTGTCCCTT-3'). The pSTU vector, directing full-length Stu2p overexpression, was created by inserting the PCR purified product, following the Qiaquick (QIAGEN, Valencia, CA) method, into the HpaI site of the pCM189 vector (GARI et al. 1997 ). The constructs were verified by sequencing, using as primers pCM2463 (5'-ACGCAAACACAAATACACACAC-3') and pCM2587 (5'-AGGGCGTGAATGTAAGCGTGAC-3').

Detection of ß-galactosidase activity on filters:
Three days after transformation, cells were replicated onto a membrane (Hybond N, Amersham, Arlington Heights, IL) and frozen three times in liquid nitrogen for 30 sec. Filters were incubated at 30° in the presence of 30 µg/ml 5-bromo-4-chloro-3 indol ß-D-galactoside (X-gal).

Quantification of readthrough efficiency:
The reporter plasmid pAC-TMV bears the same CAA UAG CAA UUA readthrough sequence as the YONX strain, but inserted between the lacZ and the luc reporter coding sequences. This vector, together with each of the candidate plasmids, was introduced into the Y349 strain by the lithium acetate method. At least three independent transformants, cultivated in the same conditions, were assayed. Cells were broken using acid-washed glass beads, as described (STAHL et al. 1995 ). Luciferase and ß-galactosidase activities were assayed in the same crude extract. Readthrough efficiency is estimated by the ratio of luciferase activity to ß-galactosidase activity. Clones were classified as suppressors (named UP) if the readthrough efficiency showed a 15% increase or more, or as antisuppressors (named DOWN) if readthrough efficiency decreased at least 15%, in comparison to wild-type readthrough efficiency. Throughout the course of the work, only figures presenting <10% standard error in three independent experiments were retained.

Identification of the candidate genes:
For each of the candidate clones, release of plasmid DNA from yeast was performed as described in HOFFMAN and WINSTON 1987 and used to transform Escherichia coli strain DH5. DNA was extracted from transformants and a restriction analysis was first performed to control for major DNA rearrangements. The boundaries of the insert were then sequenced using -21M13 and M13-reverse primers, for the clones showing no major rearrangements. This permitted us to determine the coordinates of the genomic region and thus identify the ORFs and genes present on the insert, by comparison with data obtained from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Initial screening:
In the YONX strain, the ASN1 gene is interrupted by a lacZ gene, which has an in-frame stop codon in the TMV leaky context (SKUZESKI et al. 1991 ; FEARON et al. 1994 ). This stop codon directs 25% of readthrough in a wild-type genetic background (BONETTI et al. 1995 ; STAHL et al. 1995 ) that leads to a low ß-galactosidase expression. This permits the isolation of both suppressors and antisuppressors in the same experiment. To identify genes whose overexpression interferes with translation termination, YONX cells were transformed with a multicopy S. cerevisiae genomic library.

YONX colonies displayed a moderate blue color in the presence of X-gal. In pilot experiments, the coloration appeared after 15 min at 30° for dark blue colonies, after 30 min for wild-type colonies, and after 45 min for light blue colonies. Due to the stability of ß-galactosidase, there is a high accumulation of the X-gal product. After 30 min of incubation, wild-type colonies presented the same color as dark blue clones, and after 1 hr all colonies displayed the same color intensity. The time necessary for the appearance of the color was therefore more reliable than the color intensity at a given time and was subsequently used to identify clones with differing ß-galactosidase activities.

Of 36,000 transformants, corresponding to 16 genome equivalents, 19 displayed a plasmid-dependent increase in ß-galactosidase activity, and 91 displayed a plasmid-dependent decrease in ß-galactosidase activity (Fig 2). DNA extraction and subsequent restriction analysis of the 110 candidate vectors showed that the average length of the fragments was 6 kb. Nineteen recombinant plasmids presented an abnormal restriction pattern and were not analyzed further.

View larger version (25K): In this window In a new window Download PPT slide   Figure 2. Schematic representation of the genetic screen. During the first round of screening, a multicopy genomic DNA library was used to isolate vectors modifying ß-galactosidase activity in the YONX strain. In the second round, the reporter plasmid pAC-TMV was used to identify clones specifically affecting translational readthrough.

Secondary screen:
One may anticipate that overexpression of many factors might modify ß-galactosidase activity. Among these, some could change the mRNA transcription rate of the ASN1 promoter, the mRNA or protein stability, the efficiency of translation initiation, etc. While it would be interesting to further investigate some of these factors, they do not affect the translation termination process. To identify clones directly involved in translation termination, we used an independent reporter system (pAC vectors) permitting the specific quantification of translation termination efficiency in the presence or absence of the candidate plasmids.

The pAC vectors allow quantification of readthrough (STAHL et al. 1995 ; BIDOU et al. 2000 ), without interference from other levels of control, since they carry a dual lacZ-luc reporter gene in which the ß-galactosidase activity serves as an internal control of expression from the same mRNA. In these conditions, the ratio of luciferase to ß-galactosidase depends solely on readthrough efficiency. We used the pAC-TMV vector, bearing the sequence present in the YONX strain (CAA UAG CAA UUA), inserted at the lacZ-luc junction. Each of the 91 candidate vectors was cotransformed, with the pAC-TMV vector, into the wild-type Y349 strain. For each cotransformation, three independent clones were analyzed. Vectors allowing a modification of readthrough frequency of 15% or more were further analyzed (Fig 2 and Fig 3). Eleven DOWN and 12 UP independent genomic clones were obtained after this second round of screening.

View larger version (22K): In this window In a new window Download PPT slide   Figure 3. Identification of clones modulating readthrough efficiency. The Y349 strain was cotransformed by the pAC-TMV reporter plasmid together with each of the vectors isolated in the first screen. Values on the y-axis correspond to the variation of readthrough frequency observed between a strain carrying a candidate plasmid compared to a strain carrying a control plasmid. Each value is the mean of at least two independent measurements, with at least three independent transformants. Standard deviation for each value is indicated by the hatched box.

For each of these candidates, sequencing of the fragment boundaries was performed, allowing identification of the genomic fragment overexpressed and the genes concerned. The complete list of the genes found on these 23 clones is presented in Table 2.

Associated phenotypes:
Previous studies have reported several secondary phenotypes possibly associated with suppressor or antisuppressor mutations (SINGH 1977 ; TER-AVANESYAN et al. 1982 ; WAKEM and SHERMAN 1990 ; ONO et al. 1991 ; MURGOLA et al. 1995 ). To determine whether or not the overexpression of candidate clones had pleiotropic effects, we examined sensitivity to three different agents: temperature, osmotic pressure, and the aminoglycoside antibiotic G418.

We first examined the growth rate at 37° and 15°. No difference was detected, either for strains transformed with a parental vector having no effect on translation termination efficiency, or for strains carrying any of the candidate vectors (data not shown).

We then controlled growth in high-osmolarity conditions. The range of ethylene-glycol concentration inhibiting growth was first determined for the wild-type Y349 strain transformed by a control pFL44L plasmid. A concentration of 2.5 M induced extreme growth inhibition. We chose a concentration of 2 M to identify osmotic pressure-hypersensitive clones. Among the 23 clones, 2 displayed high sensitivity (clones 51 and 408) and 4 displayed moderate sensitivity (clones 251, 432, 468, and 587; see Table 3 and Fig 4).

View larger version (81K): In this window In a new window Download PPT slide   Figure 4. Secondary phenotypes. Exponentially growing cells were serially diluted (3 x 106, 6 x 105, 1.2 x 105, 2.4 x 104, and 4.8 x 103 cells), spotted on the indicated media, and incubated at 30° for 3 days (see MATERIALS AND METHODS). The sensitivity of each clone is observed at the highest dilutions.

Finally, we employed the aminoglycoside G418. This antibiotic has been described as dramatically increasing translation termination readthrough (MANUVAKHOVA et al. 2000 ). The growth rate inhibition was examined at a concentration of 500 µg/ml. Among 23 clones tested, 3 displayed a very high growth rate inhibition (clones 29, 51, and 468) and 5 displayed a moderate growth rate inhibition (clones 96, 232, 251, 432, and 527; see Table 3 and Fig 4).

These experiments identified four clones displaying hypersensitivity to both G418 and ethylene glycol. Other clones were sensitive either to G418 or to ethylene glycol (Table 3). Thirteen clones displayed none of the secondary phenotypes sought.

Identification of candidate genes:
Since each genomic fragment bore several genes and ORFs, additional characterization was necessary to identify the sequence whose overexpression is responsible for the observed phenotype.

For further characterization, we chose clones with high sensitivity to G418 and ethylene glycol and for which at least one ORF present on the insert displayed a known function. We also retained two vectors (clones 96 and 232) carrying overlapping genomic fragments.

Vector 51:

Three ORFs and one tRNA gene are present on the 6.2-kb genomic DNA fragment. The best candidate is the gene encoding tRNA_UUG^Gln, since it has been shown that tRNA^Gln can act as a natural suppressor in eukaryotic cells (WEISS et al. 1987 ; FENG et al. 1990 ; KUCHINO and MURAMATSU 1996 ). This gene was subcloned in parental pFL44L and the resulting vector (ptRNA) was cotransformed with the pAC-TMV vector into the Y349 strain. The modification of readthrough efficiency obtained with ptRNA was identical to that previously obtained with the original vector 51 (Fig 5). Thus, overexpression of tRNA^Gln increases readthrough efficiency from 25% to >33%.

View larger version (25K): In this window In a new window Download PPT slide   Figure 5. Identification of candidate genes. Subcloning experiments were performed for vectors 468, 51, and 96/232. Results are the mean of at least three independent measurements. The y-axis corresponds to the readthrough increment (i.e., 10 means that the readthrough efficiency increases from 25 to 35%).

Vector 468:

Three complete ORFs are present on the 4-kb genomic fragment. One is the unknown ORF YPL233w; two are known genes: VMA11 encodes a proteolipid subunit of the vacuolar ATPase, an H[+]-translocating ATPase that hydrolyzes ATP to ADP and Pi, to drive protons across the vacuolar membrane into the lumen (HIRATA et al. 1997 ), while SSO1 encodes a syntaxin homolog (t-SNARE) involved in vesicle transport between the Golgi apparatus and the plasma membrane. None of these genes has been previously shown to be active in translation termination.

To identify the gene or ORF responsible for the readthrough effect, this region was subcloned in two fragments into an empty pFL44L vector, in the same orientation as that of the parental clone. The first bore VMA11 and YPL233w sequences (pVMA11) and the second, YPL233w and SSO1 (p468). These vectors were used to transform the Y349 strain, along with the pAC-TMV vector, in order to quantify translation termination efficiency. The results are shown in Fig 5. Only the p468 vector reproduced the effect obtained with the parental vector. We thus conclude that the gene responsible for the translation termination phenotype is SSO1.

Vectors 96 and 232:

These vectors have overlapping genomic DNA fragments of 6.3 and 7.8 kb. The STU2 gene, which encodes a microtubule-binding protein, is the only complete gene present in both fragments. However, the accumulation of a truncated gene product may induce its association with a new set of molecules involved in translation termination, even though it normally has no role in this process. To determine whether the overexpression of Stu2p is responsible for the readthrough increase, we cloned the STU2 open reading frame under the control of the strong CYC promoter in the pCM189 plasmid (GARI et al. 1997 ), yielding the pSTU plasmid. Cotransformation of pSTU and pAC-TMV constructs in the Y349 strain reproduced the effect obtained with the parental vectors (Fig 5).

Effect of STU2 overexpression on different readthrough sites:
To determine whether STU2 overexpression increased readthrough on sequences other than the TMV motif used in the screen, we exploited a set of vectors that carry different stop codons in different contexts and drive various readthrough levels. Results shown in Fig 6 demonstrate that, although the range of the effect varies depending on the target, the readthrough level was consistently increased. Only very strong stop codons (vectors 71, 72, and 73) did not seem to respond to STU2 overexpression, but the very low readthrough levels obtained with these vectors (i.e., <1%) would hardly allow observation of significant variations.

View larger version (24K): In this window In a new window Download PPT slide   Figure 6. Effect of STU2 overexpression on different targets. The name and the stop codon present on these targets are given in the x-axis. Readthrough levels were measured in the presence or in the absence of the STU2 overexpression vector. Readthrough frequency is defined as the ratio of luciferase activity to ß-galactosidase activity. The ratio of luciferase activity to ß-galactosidase from an in-frame control plasmid was used as reference. Values are the mean of at least three independent experiments. The sequences of the oligonucleotides used to construct the different reporters are given in Table 1.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In eukaryotes, the mechanisms of translation termination remain to be completely elucidated. In particular, the precise role of eRF3 remains obscure, and almost nothing is known about the ultimate events linked to ribosomal dissociation. Each of these steps is a potential target for gene expression control. To date, interaction with UPF components remains the sole mechanism supported by genetic and functional evidence and clearly modifies the termination activity of RFs (BIDOU et al. 2000 ; MADERAZO et al. 2000 ; WANG et al. 2001 ). The aim of our study was to identify new partners involved in translation termination, in the hope of obtaining more information on the mechanisms involved.

We chose a dosage-suppressor approach to isolate candidate factors interacting with the termination translation machinery. Recently, a similar approach was used by Ter-Avanesyan and collaborators to isolate the Itt1p protein that inhibits translation termination in S. cerevisiae (URAKOV et al. 2001 ). The use of a lacZ reporter gene carrying a leaky stop codon promoting 25% readthrough allowed us to isolate and identify genomic regions whose overexpression provokes either an increase or a decrease in translation termination efficiency.

In an initial screen, we identified 110 clones inducing a modification of ß-galactosidase activity among 36,000 colonies. A second screen, based on a dual-reporter system specifically assessing modification of readthrough levels, selected 23 clones, which were further analyzed. A third screen identified secondary phenotypes often associated with suppressor and antisuppressor mutations: temperature, G418, or hyperosmotic sensitivity (SINGH 1977 ). The genes present on each genomic DNA fragment of the 23 clones were identified by sequencing the fragment boundaries. For several candidate clones, subcloning determined the gene responsible for the translation termination phenotype.

Clone 51 displays a high sensitivity to both osmotic pressure and G418. The presence of this vector results in an increase of readthrough efficiency of 33%. Subcloning demonstrated that the tRNA^Gln is responsible for this increase. As described (WEISS et al. 1987 ), overexpression of a tRNA^Gln allows stop codon suppression in S. cerevisiae. The simplest explanation for the role of this tRNA is that it acts directly as a natural suppressor of the UAG stop codon when overexpressed. However, we cannot exclude an indirect role, since this tRNA also recognizes glutamine codons surrounding the stop codon present in our reporter system. In any case, this clone clearly validates our screening procedure.

Clone 468 confers a moderate sensitivity to osmotic pressure and a high sensitivity to G418. It induces a 58% increase in readthrough frequency. This effect is one of the strongest observed over the course of these experiments. Among the three genes borne by this plasmid, subcloning experiments indicated that the SSO1 gene is responsible for the phenotype. This protein encodes a syntaxin homolog (t-SNARE) involved in vesicle transport and has no known relation with the translation termination process. Previous work has demonstrated that any defect in the secretory pathway leads to a rapid repression of both rRNA and ribosomal proteins (MIZUTA and WARNER 1994 ) and it is known that the overexpression of the SSO1 gene enhances the secretory pathway (RUOHONEN et al. 1997 ). Following this logic, we postulate that under these conditions, translation efficiency will be enhanced, increasing the number of ribosomes on the mRNA, which may in turn affect the translational termination process. Several other proteins involved in cellular transport are present in genomic inserts and may act similarly on translation termination. Future experiments will be necessary to define precisely which proteins are involved in this intriguing relation and to understand how these two pathways are connected.

Clones 96 and 232 show the same sensitivity to G418 and no hypersensitivity to osmotic pressure. They carry overlapping genomic DNA fragments that induce an increase of 35–39% in readthrough efficiency and we demonstrated that STU2 is actually responsible for the phenotype. STU2 encodes a microtubule-binding protein, a component of the spindle pole body (WANG and HUFFAKER 1997 ; SEVERIN et al. 2001 ). Stu2p overexpression could modify translation termination efficiency by either a direct or an indirect mechanism. It should be noted that Stu2p overexpression affects translation termination efficiency at all three stop codons and in various nucleic contexts (see Fig 6).

Numerous studies have described a complex relation between the translation apparatus and the cytoskeleton (CONDEELIS 1995 ; EDMONDS et al. 1995 ; DEFRANCO et al. 1998 ; MOORE et al. 1998 ; BAILLEUL et al. 1999 ; REGULA et al. 2001 ). eEF-1A, which shares sequence similarities with eRF3, binds aminoacyl-tRNA and interacts with actin filaments (MOORE et al. 1998 ). The presence of EF-1A in centrosomes of sea urchin (HAMILL et al. 1994 ) and the ability of this protein to bind microtubules suggests that it could take part in cytoskeletal functions. In Drosophila melanogaster, depletion of a homolog of yeast Sup35p resulted in cytoskeleton defects in spermatids and in abnormal meiotic chromosome segregation (BASU et al. 1998 ). The functional link between the normal function of eRF3 in translation termination and the effect of its mutation in spermatids remains unclear. However, these results also suggest the existence of a pathway integrating the cytoskeleton and the translational machinery in eukaryotic cells, which could participate in a translational subcompartment, as suggested by Deutscher and co-workers (STAPULIONIS and DEUTSCHER 1995 ; STAPULIONIS et al. 1997 ). Our results also support this interpretation, pointing to a similar relation using an independent approach on a different model system. This relation could be explained by another important function of SUP35 and/or SUP45 genes in cytoskeletal organization, such as coordination of the cytoskeletal and translational machinery or control of chromosomal transmission, as suggested by Inge-Vechtomov (BORCHSENIUS et al. 2000 ). Although convergent observations suggest a connection between translation termination and the cytoskeleton, an indirect effect of Stu2p cannot presently be ruled out.

Several other clones, not yet completely analyzed, are also interesting. Clone 28 carries the RRN3 gene. This protein is required for efficient transcription by RNA polymerase I, which transcribes the rDNA. We subcloned the RRN3 gene in the plasmid pFL44L, but overexpression of this gene alone did not mimic the effect observed with clone 28 (data not shown). Clone 111 carries a genomic fragment of rDNA, in which only the gene encoding 5S-rRNA is complete. Although we cannot exclude that a truncated rRNA (25S or 18S) is responsible for the effect on translation termination, 5S-rRNA is our best candidate, since it has already been shown to be involved in translation accuracy. Dinman and co-workers isolated 5S-rRNA mutants deficient in the maintenance of a correct reading frame (DINMAN and WICKNER 1995 ). A number of clones also carry ORFs only without any known or predicted functions. Given the specificity of our screen, based on a reliable dual reporter, it is very likely that several new factors will emerge from the detailed analysis of these candidate vectors, in particular those whose overexpression decreases translational readthrough, since such a direct screen for antisuppressors has never been undertaken before.

Overall, our results indicate potential new links between translation termination and other cellular functions, some of which have already been shown to have functional connections with translation. Physiological and molecular studies must now be performed to elucidate the underlying mechanisms. It is also noteworthy that the screen is not saturated and could lead to the selection of additional mutants.

	FOOTNOTES

¹ Present address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom.
² Present address: Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250.
³ Present address: Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30333.
⁴ Present address: Laboratoire de Génétique Moléculaire et Cellulaire, INRA, CNRS Institut National Agronomique, 78850 Thiverval-Grignon, France.

	ACKNOWLEDGMENTS

The authors are indebted to Monique Bolotin-Fukuhara and Marguerite Picard for their constant help and support during the course of this work and to Ian Brierley for his help with the revised manuscript. This work was supported in part by the Association pour la Recherche contre le Cancer (contract 9873 to J.-P.R.). G. S. was partially supported by National Institutes of Health grant GM-29480 to Philip Farabaugh. H.L. was supported by fellowships from CNRS-K. C. Wong Foundation and Fondation pour la Recherche Médicale.

Manuscript received July 23, 2001; Accepted for publication March 21, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AALTO, M. K., H. RONNE, and S. KERANEN, 1993  Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. EMBO J. 12:4095-4104[Medline].

BAILLEUL, P. A., G. P. NEWNAM, J. N. STEENBERGEN, and Y. O. CHERNOFF, 1999  Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae. Genetics 153:81-94[Abstract/Free Full Text].

BASU, J., B. C. WILLIAMS, Z. LI, E. V. WILLIAMS, and M. L. GOLDBERG, 1998  Depletion of a Drosophila homolog of yeast Sup35p disrupts spindle assembly, chromosome segregation, and cytokinesis during male meiosis. Cell Motil. Cytoskeleton 39:286-302[Medline].

BIDOU, L., G. STAHL, I. HATIN, O. NAMY, and J. P. ROUSSET et al., 2000  Nonsense-mediated decay mutants do not affect programmed-1 frameshifting. RNA 6:952-961[Abstract].

BONETTI, B., L. W. FU, J. MOON, and D. M. BEDWELL, 1995  The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J. Mol. Biol. 251:334-345[Medline].

BONNEAUD, N., O. OZIER-KALOGEROPOULOS, G. LI, M. LABOUESSE, and L. MINVIELLE-SEBASTIA et al., 1991  A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7:609-615[Medline].

BORCHSENIUS, A. S., A. A. TCHOURIKOVA, and S. G. INGE-VECHTOMOV, 2000  Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae. Curr. Genet. 37:285-291[Medline].

CONDEELIS, J., 1995  Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem. Sci. 20:169-170[Medline].

CZAPLINSKI, K., Y. WENG, K. W. HAGAN, and S. W. PELTZ, 1995  Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. RNA 1:610-623[Abstract].

CZAPLINSKI, K., N. MAJLESI, T. BANERJEE, and S. W. PELTZ, 2000  Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency. RNA 6:730-743[Abstract].

DANG, V. D., M. VALENS, M. BOLOTIN-FUKUHARA, and B. DAIGNAN-FORNIER, 1996  Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor. Mol. Microbiol. 22:681-692[Medline].

DEFRANCO, C., M. E. CHICUREL, and H. POTTER, 1998  A general RNA-binding protein complex that includes the cytoskeleton-associated protein MAP 1A. Mol. Biol. Cell 9:1695-1708[Abstract/Free Full Text].

DINMAN, J. D. and R. B. WICKNER, 1995  5 S rRNA is involved in fidelity of translational reading frame. Genetics 141:95-105[Abstract].

EDMONDS, B. T., J. MURRAY, and J. CONDEELIS, 1995  pH regulation of the F-actin binding properties of Dictyostelium elongation factor 1 alpha. J. Biol. Chem. 270:15222-15230[Abstract/Free Full Text].

FARABAUGH, P., Q. QIAN and G. STAHL, 2000 Programmed translational frameshifting, hopping and readthrough of termination codons, pp. 741–761 in Translational Control of Gene Expression, edited by N. SONENBERG, J. W. B. HERSHEY and M. MATHEWS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

FEARON, K., V. MCCLENDON, B. BONETTI, and D. M. BEDWELL, 1994  Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. J. Biol. Chem. 269:17802-17808[Abstract/Free Full Text].

FENG, Y. X., T. D. COPELAND, S. OROSZLAN, A. REIN, and J. G. LEVIN, 1990  Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus. Proc. Natl. Acad. Sci. USA 87:8860-8863[Abstract/Free Full Text].

GARI, E., L. PIEDRAFITA, M. ALDEA, and E. HERRERO, 1997  A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13:837-848[Medline].

HAMILL, D., J. DAVIS, J. DRAWBRIDGE, and K. A. SUPRENANT, 1994  Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos. J. Cell Biol. 127:973-984[Abstract/Free Full Text].

HIRATA, R., L. A. GRAHAM, A. TAKATSUKI, T. H. STEVENS, and Y. ANRAKU, 1997  VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase. J. Biol. Chem. 272:4795-4803[Abstract/Free Full Text].

HOFFMAN, C. S. and F. WINSTON, 1987  A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267-272[Medline].

HOSHINO, S., M. IMAI, T. KOBAYASHI, N. UCHIDA, and T. KATADA, 1999  The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-Poly (A) tail of mRNA. Direct association of erf3/GSPT with polyadenylate-binding protein. J. Biol. Chem. 274:16677-16680[Abstract/Free Full Text].

ITO, H., Y. FUKUDA, K. MURATA, and A. KIMURA, 1983  Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168[Abstract/Free Full Text].

KISSELEV, L. L. and L. Y. FROLOVA, 1999  Termination of translation in eukaryotes: new results and new hypotheses. Biochemistry 64:8-16[Medline].

KUCHINO, Y. and T. MURAMATSU, 1996  Nonsense suppression in mammalian cells. Biochimie 78:1007-1015[Medline].

LEEDS, P., S. W. PELTZ, A. JACOBSON, and M. R. CULBERTSON, 1991  The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 5:2303-2314[Abstract/Free Full Text].

MADERAZO, A. B., F. HE, D. A. MANGUS, and A. JACOBSON, 2000  Upf1p control of nonsense mRNA translation is regulated by Nmd2p and Upf3p. Mol. Cell. Biol. 20:4591-4603[Abstract/Free Full Text].

MANUVAKHOVA, M., K. KEELING, and D. M. BEDWELL, 2000  Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6:1044-1055[Abstract].

MIZUTA, K. and J. R. WARNER, 1994  Continued functioning of the secretory pathway is essential for ribosome synthesis. Mol. Cell. Biol. 14:2493-2502[Abstract/Free Full Text].

MOORE, R. C., N. A. DURSO, and R. J. CYR, 1998  Elongation factor-1alpha stabilizes microtubules in a calcium/calmodulin-dependent manner. Cell Motil. Cytoskeleton 41:168-180[Medline].

MURGOLA, E. J., F. T. PAGEL, K. A. HIJAZI, A. L. ARKOV, and W. XU et al., 1995  Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA. Biochem. Cell Biol. 73:925-931[Medline].

NAMY, O., G. DUCHATEAU-NGUYEN, and J.-P. ROUSSET, 2002  Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol. Microbiol. 43:641-652[Medline].

ONO, B., Y. O. CHERNOFF, Y. ISHINO-ARAO, N. YAMAGISHI, and S. SHINODA et al., 1991  Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae. Curr. Genet. 19:243-248[Medline].

REGULA, J. T., G. BOGUTH, A. GORG, J. HEGERMANN, and F. MAYER et al., 2001  Defining the mycoplasma "cytoskeleton": the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry. Microbiology 147:1045-1057[Abstract/Free Full Text].

RUOHONEN, L., J. TOIKKANEN, V. TIEAHO, M. OUTOLA, and H. SODERLUND et al., 1997  Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery. Yeast 13:337-351[Medline].

SEVERIN, F., B. HABERMANN, T. HUFFAKER, and T. HYMAN, 2001  Stu2 promotes mitotic spindle elongation in anaphase. J. Cell Biol. 153:435-442[Abstract/Free Full Text].

SHUMOV, N. N., K. V. VOLKOV, and L. N. MIRONOVA, 2000  Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast. Genetika 36:644-650[Medline].

SINGH, A., 1977  Nonsense suppressors of yeast cause osmotic-sensitive growth. Proc. Natl. Acad. Sci. USA 74:305-309[Abstract/Free Full Text].

SKUZESKI, J. M., L. M. NICHOLS, R. F. GESTELAND, and J. F. ATKINS, 1991  The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. J. Mol. Biol. 218:365-373[Medline].

STAHL, G., L. BIDOU, J. P. ROUSSET, and M. CASSAN, 1995  Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells. Nucleic Acids Res. 23:1557-1560[Abstract/Free Full Text].

STANSFIELD, I., K. M. JONES, V. V. KUSHNIROV, A. R. DAGKESAMANSKAYA, and A. I. POZNYAKOVSKI et al., 1995  The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 14:4365-4373[Medline].

STAPULIONIS, R. and M. P. DEUTSCHER, 1995  A channeled tRNA cycle during mammalian protein synthesis. Proc. Natl. Acad. Sci. USA 92:7158-7161[Abstract/Free Full Text].

STAPULIONIS, R., S. KOLLI, and M. P. DEUTSCHER, 1997  Efficient mammalian protein synthesis requires an intact F-actin system. J. Biol. Chem. 272:24980-24986[Abstract/Free Full Text].

TER-AVANESYAN, M. D., J. ZIMMERMANN, S. G. INGE-VECHTOMOV, A. B. SUDARIKOV, and V. N. SMIRNOV et al., 1982  Ribosomal recessive suppressors cause a respiratory deficiency in yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 185:319-323[Medline].

TER-AVANESYAN, M. D., A. R. DAGKESAMANSKAYA, V. V. KUSHNIROV, and V. N. SMIRNOV, 1994  The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae.. Genetics 137:671-676[Abstract].

TIKHOMIROVA, V. L. and S. G. INGE-VECHTOMOV, 1996  Sensitivity of sup35 and sup45 suppressor mutants in Saccharomyces cerevisiae to the anti-microtubule drug benomyl. Curr. Genet. 30:44-49[Medline].

URAKOV, V. N., I. A. VALOUEV, E. I. LEWITIN, S. V. PAUSHKIN, and V. S. KOSORUKOV et al., 2001  Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae. Biomed. Chromatogr. Mol. Biol. 2:9.

WAKEM, L. P. and F. SHERMAN, 1990  Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae. Genetics 124:515-522[Abstract].

WANG, P. J. and T. C. HUFFAKER, 1997  Stu2p: A microtubule-binding protein that is an essential component of the yeast spindle pole body. J. Cell Biol. 139:1271-1280[Abstract/Free Full Text].

WANG, W., K. CZAPLINSKI, Y. RAO, and S. W. PELTZ, 2001  The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts. EMBO J. 20:880-890[Medline].

WEISS, W. A., I. EDELMAN, M. R. CULBERTSON, and E. C. FRIEDBERG, 1987  Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 84:8031-8034[Abstract/Free Full Text].

ZHOURAVLEVA, G., L. FROLOVA, X. LE GOFF, R. LE GUELLEC, and S. INGE-VECHTOMOV et al., 1995  Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14:4065-4072[Medline].

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