Copyright Ó 2007 by the Genetics Society of America DOI: 10.1534/genetics.107.070771 Fine-Tuning of Translation Termination Efficiency in Saccharomyces cerevisiae Involves Two Factors in Close Proximity to the Exit Tunnel of the Ribosome Isabelle Hatin,*,†,1 Ce´line Fabret,*,† Olivier Namy,*,† Wayne A. Decatur‡ and Jean-Pierre Rousset*,† *IGM, Universite´ Paris-Sud, UMR 8621, F91405 Orsay, France, †CNRS, F91405 Orsay, France and ‡Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Manuscript received January 11, 2007 Accepted for publication April 27, 2007 ABSTRACT In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we con- structed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level. RANSLATION termination is the step that liber- somal P-site (Mottagui-Tabar and Isaksson 1998), T ates the newly synthesized polypeptide from the (ii) the mRNA structure shape due to the nucleotide se- ribosome before recycling the translational machinery. quence at the P site that could alter decoding through Three triplets—UAA, UAG (Weigert and Garen 1965), distortion of the ribosome structure (Tork et al. 2004), and UGA (Brenner et al. 1967)—were identified as and (iii) the chemical property of the amino acid at nonsense stop codons and shown to serve in vitro as sig- the penultimate position. Previous analyses have shown nals for the release of polypeptide from the ribosome that the nucleotides 39 of the stop have a predominant (Takanami and Yan 1965). The misincorporation of an role on readthrough efficiency and that the 59 context amino acid at the stop codon occurs at a frequency of effect is dependent on the 39 context (Skuzeski et al. 10À4 and is called readthrough. The efficiency of this 1991; Bonetti et al. 1995; Howard et al. 1996; Mottagui- termination is modulated by cis and trans factors. In Tabar and Isaksson 1998; Cassan and Rousset 2001; general, release factors efficiently recognize the termi- Namy et al. 2001). In particular, the nucleotide imme- nation codons, but in certain instances, near-cognate diately following the stop is highly biased in prokaryotes transfer RNAs (tRNAs) overcompete and lead to read- and eukaryotes and it has been proposed that the stop through. tRNA decoding of a stop codon occurs more signal could involve four nucleotides (nt) (Brown et al. frequently when the stop codon is surrounded by a context 1990). Several studies have pointed to at least three nt that modifies the competition for stop codon recognition upstream and six nt downstream of the stop to be in- between a release factor and near-cognate tRNA (Salser volved in determining readthrough efficiency (Bonetti 1969; Fluck and Epstein 1980; Engelberg-Kulka 1981). et al. 1995; Namy et al. 2001). Aminoglycosides can in- In Saccharomyces cerevisiae,both59 and 39 sequences play a crease readthrough and have been shown to suppress role in translation termination (Bonetti et al. 1995; premature stop mutations in several animal and cul- Namy et al. 2001; Tork et al. 2004). Several studies point tured cell models (Bedwell et al. 1997; Barton-Davis to different elements that could be involved in the 59 et al. 1999; Manuvakhova et al. 2000; Bidou et al. 2004). effect in S. cerevisiae: (i) the tRNA located on the ribo- These observations have opened the possibility of treat- ing patients who bear nonsense mutations with amino- glycoside antibiotics to express full-length protein. Given 1Corresponding author: Institut de Ge´ne´tique et Microbiologie, Baˆtiment 400, Universite´ Paris-Sud, F91405 Orsay, France. the numerous human diseases caused by nonsense mu- E-mail: [email protected] tation (Krawczak et al. 2000), it is thus imperative to Genetics 177: 1527–1537 (November 2007) 1528 I. Hatin et al. determine the precise mechanism of translation termi- interaction of eRF1 with PABp has also been shown in nation in eukaryotes. Xenopus and human cells (Cosson et al. 2002) and could In eukaryotic cells, termination necessitates the re- help recycling of translational components. In addition, cruitment of the release factors eRF1 and eRF3 by the Itt1p (Urakov et al. 2001) and PP2A (Andjelkovic et al. ribosomal machinery at the A-site. eRF1 is involved in 1996) have been described to interact with eRF1, but stop codon recognition but fully efficient termination without clue on the mechanism of translational termi- needs interaction with the GTPase eRF3. In 1994, Frolova nation mediated by these interactions. Several observa- and coworkers showed that SUP45 protein of S. cerevisiae tions also suggest a link between termination and the belongs to a highly conserved eukaryotic protein family cytoskeleton. Sla1p is involved in the cytoskeleton and and corresponds most likely to the yeast eRF1 (Frolova has been found to interact with the N-terminal domain et al. 1994). That assignment was subsequently experimen- of eRF3 (Bailleul et al. 1999). Actin mutants have been tally demonstrated by Stansfield et al. (1995b). eRF1 associated with increased readthrough on the UAA stop comprises three domains: the N-terminal domain involved codon (Kandl et al. 2002), and a microtubule binding in stop codon recognition (Bertram et al. 2000; Song et al. protein of the spindle pole body Stu2p has been iden- 2000; Chavatte et al. 2001) and the M domain that con- tified in a genetic screen for factors modulating trans- tains a GGQ motif highly conserved throughout evolution lational termination efficiency (Namy et al. 2002). (Frolova et al. 1999), which is responsible for peptidyl Apart from the above-mentioned proteins, one can transferase hydrolytic activity. These two domains form envision that other factors able to modulate the termina- the functionally active ‘‘core’’ (Frolova et al. 2000). The tion process remain to be discovered. Indeed, overexpres- third domain in eRF1, the C-terminal domain, is involved sion of yeast eRF factors, Sup45p and Sup35p, increases in the interaction with the protein phosphatase PP2A translational termination efficiency no more than 2.6- (Andjelkovic et al. 1996) and with eRF3 (Stansfield et al. fold (Stansfield et al. 1995b; Williams et al. 2004). To 1995b; Zhouravleva et al. 1995). eRF3, encoded by SUP35 identify antisuppressors limiting near-cognate, tRNA- in S. cerevisiae,ismadeupofthreedomains.TheN- mediated suppression, we developed a screen for factors terminal and M domains are not essential for viability and that would increase translational termination when over- termination (Ter-Avanesyan et al. 1993). In S. cerevisiae, expressed (multicopy antisuppressors). For this purpose, the N terminus is asparagine and glutamine rich and un- we used a strain that carries an allele of the ADE2 gene, derlies the conformational changes of eRF3 to proteinase- interrupted by an in-frame UAG stop codon surrounded resistant aggregates, leading to the ½PSI1 phenotype (see by sequences known to promote a readthrough level review in Patino et al. 1996; Paushkin et al. 1996; high enough to obtain white colonies. We screened for Chernoff 2001; Cosson et al. 2002). ½PSI1 cells present candidate DNA fragments able to confer a red color to a defect in translation termination characterized by an the colonies. Among those, SSB1 and snR18 sequences omnipotent nonsense suppression phenotype (Liebman were found repeatedly and have been shown to actually and Sherman 1979). The C-terminal domain carries decrease the readthrough level. The mechanism of GTPase activity (Frolova et al. 1996), which is essential SSB1-induced readthrough decrease has been further for viability and termination and interacts with eRF1 characterized. and Upf1 (Stansfield et al. 1995b; Weng et al. 1996; Czaplinski et al. 1998). Recently, Salas-Marco and edwell B (2004) showed that eRF3 mutants with a MATERIALS AND METHODS reduced GTPase activity lead to a decreased translation termination efficiency. Recent results suggest that a Yeast strains and media: The S. cerevisiae strains used for this work are OL556 (MATa/MATa, cdc25-5/cdc25-5 his3/his3 leu2/ stable interaction between eRF1 and the stop codon in oy arcotte the A-site stimulates eRF3 GTP hydrolysis, which leads to leu2 trp1/TRP1 rca1/rca1 ura3/ura3)(B -M et al. 1996), 74D694 (MATa ade1-14 trp1-289 leu2-3,112 his3-200 ura3-52) efficient release of the polypeptide from the ribosome 1 erkatch a alas arco edwell lkalaeva ½psiÀ; ½psi (D et al. 1998), MT557/3b (MAT ade2-1 by eRF1 (S -M and B 2004; A sup45-2 leu2-3,112 ura3-1 his5-2)(Stansfield et al. 1995a), and et al. 2006). In spite of genetic, biochemical, and FS1 (MATa, ade2-592 lys2-201 leu2-3,112 his3-200 ura3-52) crystallographic analyses of eRF1 and eRF3, questions (Namy et al. 2001). about the translational termination mechanism remain. The modified FS1strain used in the screen was constructed In particular, several factors have been demonstrated to as follows: From the ADE2 gene and its promoter cloned in a interact with the termination process, either directly centromeric URA3 vector (pFL38), a readthrough sequence through contacts with release factors or indirectly,as dem- derived from tobacco mosaic virus (TMV) (GGAACACAA TAGCAGTTACAG) was cloned in the unique HpaI restriction onstrated by genetic experiments.