- Research article
- Open Access
Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae
© Chabelskaya et al; licensee BioMed Central Ltd. 2007
Received: 13 December 2006
Accepted: 16 August 2007
Published: 16 August 2007
The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.
In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Δ double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants.
This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.
Two translation termination factors, eRF1 and eRF3, participate in termination of protein synthesis in eukaryotes (reviewed in ). In S. cerevisiae they are encoded by SUP45 and SUP35, respectively (reviewed in ). In eukaryotes, a single factor, eRF1 (Sup45 in yeast), decodes all three stop codons, while eRF3 (Sup35 in yeast) stimulates termination through a GTP-dependent mechanism by forming a complex with eRF1.
Eukaryotic cells possess a mechanism known as nonsense-mediated mRNA decay (NMD) to recognize and degrade mRNA molecules that contain premature termination codon (PTC) (reviewed in [3–5]). The NMD process is mediated by the trans- acting factors Upf1, Upf2 and Upf3 [6–11], all of which directly interact with eRF3; while only Upf1 interacts with eRF1 [12, 13]. Using in vitro competition experiments, it has been demonstrated that Upf2, Upf3 and eRF1 actually compete with each other for binding to eRF3 . Deletion of any one of the three UPF genes selectively stabilizes mRNAs that are degraded by the NMD pathway without affecting other mRNAs [6, 7, 9–11]. Genetic studies have shown that Upf1, Upf2, and Upf3 act as obligate partners in the NMD pathway; this means that NMD only occurs when all components are present (reviewed in [14, 15]). Mutations or deletions of UPF genes lead to an increased frequency of nonsense suppression at termination codons in a variety of yeast genes (reviewed in ). A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability and not only causes a defect in translation termination but also slows normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation . Taken together these results suggest a direct link between the termination complex and the mRNA stability.
Both eRF1 and eRF3 are essential for viability of yeast cells and deletion of the C-terminal part of each protein separately lead to lethality (reviewed in ). Nonsense sup45 mutations have been obtained in the presence of SUQ5 suppressor tRNA . However, we have isolated non-lethal nonsense mutations in the SUP45 gene of S. cerevisiae which lead to decreased level of eRF1 . Nonsense mutations were also obtained in the SUP35 gene [19, 20]. Here, we show that sup45 nonsense and missense mutations have an inhibitory effect on NMD. Our observation that loss of Upf1 suppresses many of the pleiotropic phenotypes caused by mutations in SUP45 allowed us to discuss the role of the Upf complex in translation termination.
The sup45 mutations cause a general decrease in the efficiency of NMD
In previous work, we have isolated non-lethal nonsense and missense mutations in the essential SUP45 gene of S. cerevisiae which lead to a high level of suppression . Since a direct link between the termination complex and the mRNA stability was proposed, we examined the efficiency of NMD in these mutants by testing whether a decrease in eRF1 level will lead to accumulation of PTC-containing transcripts.
Nonsense mutations sequenced in the present work
Putative destabilizing motifa
A AA→T AA
311ca TTGAT t TT a agttgtccaggt TCGATG a TTC a
TT A→TA A
C AG→T AG
436gtTTGAT t C agaagcaggtgggacaggtgaactT t T g GAT tggaacTCGAT t TCT gactgggttggaagg
Increased viability of sup45 nonsense mutants in the absence of UPF1
Previously, we have shown that sup45 nonsense mutants are viable in different genetic backgrounds . However, the efficiency of plasmid shuffle was significantly lower in the case of mutant sup45-n alleles compared to plasmid bearing wild-type SUP45 gene  indicating that sup45-n mutations imperfectly replace SUP45. To assess the effects of double sup45 upf1 mutations on viability of corresponding strains we performed plasmid shuffle analysis using strains bearing single sup45 mutations or sup45 in combination with upf1Δ (see Materials and Methods).
Two nonsense mutations resulting in different stop codons (sup45-102 (UAA) and sup45-107 (UGA)) and one missense mutation (sup45-103) were used to perform plasmid shuffle experiments. Two isogenic yeast strains 1A-D1628 (sup45Δ pRS316/SUP45) and 1-1A-D1628 (sup45Δ upf1Δ pRS316/SUP45) were transformed with the pRS315 plasmids bearing the wild-type SUP45 gene or different sup45 mutations. Transformants were then subjected to plasmid shuffle analysis to verify whether strains containing the sup45 alleles could lose the plasmid carrying the wild-type gene. In the sup45Δ strain, all transformants were able to grow in the presence of 5-FOA, indicating that all tested mutations can replace wild-type SUP45. However, as previously described , plasmid shuffle was less efficient with sup45 mutations than with wild-type SUP45. Surprisingly, introduction of upf1Δ mutation lead to increased viability of sup45 mutants (Fig. 3, 5-FOA). We did not observe difference in growth between wild-type and sup45 mutants on medium selective for both plasmids (Fig. 3, -L-U). In order to check that deletion of UPF1 does not lead to higher production of eRF1 protein in double sup45 upf1Δ mutants which could explain the increased viability of these mutants, we analyzed eRF1 protein level by western blot. As shown in figure 3B, deletion of UPF1 does not affect the level of eRF1 protein in sup45 mutants.
Deletion of the UPF1 gene suppresses several sup45 phenotypes
Defects of NMD in double mutants sup45 upf1 Δ
To compare the effects of single sup45 mutations and the sup45 upf1Δ double mutations on the efficiency of suppression, we replica plated the same transformants that were used for Nothern blots analysis (Fig. 5A) on adenine deprived medium. As shown in another genetic background (Fig. 4C), deletion of UPF1 does not promote suppression of ade1-14 mutation (Fig. 5C) and the combined effects of sup45 mutations and upf1Δ promote increase in the suppression efficiency compared with sup45 mutations alone. As shown in figure 5D, allosuppression of ade1-14 by deletion of UPF1 is not the result of stabilization of ade1-14 mRNA in double sup45 upf1Δ mutants. We also showed that deletion of UPF1 does not affect sup45-n mRNAs and eRF1 as well as eRF3 protein levels (Fig. 5D).
Deletion of either UPF2 or UPF3 increases viability of sup45 nonsense mutants
In the present work, we have shown that nonsense and missense mutations in the SUP45 gene lead to stabilization of PTC-containing mRNAs degraded by NMD. This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.
The CYH2 pre-mRNA which contains a premature termination codon was previously shown to be degraded by NMD pathway . We also identified that NMD affects accumulation of his7-1 mRNA. A single A→T mutation in this allele leads to change of codon 229 for UAA. In addition, two imperfect putative DSE are found downstream of this premature stop codon. Using a upf1Δ strain, we demonstrated that the his7-1 transcript is possibly under control of NMD pathway. In order to answer if changes in transcription of HIS7 gene could account for accumulation of his7-1 mRNA, we examined the mRNA level of wild-type HIS7 mRNA in sup45-n mutants and upf1Δ mutants. Deletion of upf1 as well as sup45 mutations leads to accumulation of his7-1 mRNA but do not affect mRNA level of wild-type HIS7 mRNA. Accordingly, a genome-wide analysis performed in strains depleted for NMD showed that wild-type HIS7 mRNA (and also ADE1 and LYS9 mRNAs) is not affected in strains deleted for upf1 .
Here, we demonstrate that accumulation of his7-1 and CYH2 precursor mRNAs in cells bearing sup45 mutations was much higher than that observed in the wild-type strain. But, sup45 mutations do not promote accumulation of other nonsense-containing transcripts, such as ade1-14 or lys9-A21, despite efficient suppression of these mutations, as well as his7-1 [18, 22]. This result indicates that simply increasing read-through efficiency does not result in a general increase in the abundance of PTC-containing mRNAs but that sup45 mutations specifically affect PTC-containing mRNA subjected to NMD. We observed that the abundance of his7-1 and CYH2 precursor mRNAs in cells bearing sup45 mutations was lower compared to those in the upf1Δ strain. This difference between upf1Δ and sup45 mutants could be explained by the complete absence of Upf1 protein in the upf1Δ strain leading to complete inactivation of NMD and by the presence of some functional eRF1 protein in sup45 mutants which is necessary for cell viability [18, 22]. Indeed, we previously reported that in the case of sup45-n, the level of eRF1 is decreased compared to wild-type and in the case of sup45 missense mutants the level of eRF1 is unchanged but its functionality is altered. These results demonstrate that eRF1 participates in NMD.
Recently, it was shown the importance of the second translation termination factor (eRF3) for NMD and interaction of both eRF1 and eRF3 with Upf proteins. Upf1 protein interacts with the polypeptide release factors eRF3 and eRF1 while they are still present in the ribosome-bound termination complex, providing a direct link between the termination complex and the NMD machinery [12, 13]. Both Upf2 and Upf3 interact with eRF3, but not with eRF1; and Upf2, Upf3 and eRF1 compete with each other in vitro for binding to eRF3 [12, 13]. eRF3 also interacts with poly(A)-binding protein (PABP) [27, 28], furthermore, eRF3 regulates the initiation of normal mRNA decay at the poly(A) tail-shortening step through the interaction with PABP . Thus, eRF3 can mediate normal and nonsense-mediated mRNA decay through its association with Pab1 and Upf1 and therefore was proposed as a key mediator between translation termination and NMD . Moreover, it was previously shown that a weak translation termination due to [PSI+] (a prion form of eRF3) antagonizes the effects of NMD . A first indication for a link between translation termination and NMD came from observations that decay of PTC-containing mRNAs can be antagonized by tRNAs that suppress termination . Data have shown that normal termination is distinct from premature termination and this difference is dependent upon the presence of Upf1 at the premature termination codon . Our results together with data about eRF1-Upf1 interaction [12, 13] demonstrate that eRF1 as well as eRF3 is an essential factor linking translation termination and NMD. Recognition of stop codons is a common event necessary for the two processes. Since it is established that eRF1 plays a crucial role in translation termination by directly recognizing stop codons (reviewed in ), eRF1 could have an identical function in NMD by recognition of PTC.
We observed that the combination of upf1Δ and sup45-n mutations leads to an increase in CYH2 precursor mRNA abundance that was higher than in upf1Δ and sup45-n single mutants. A similar additive effect on stabilization of nonsense-containing mRNA was shown for combination of upf1Δ and [PSI+] . Therefore, a possible explanation for this additive effect of upf1Δ and sup45-n mutations could be that eRF1 is required for both normal and nonsense-mediated mRNA decay, as it was shown for eRF3 .
It has been shown that mutation in eRF3 which impairs eRF3 binding to eRF1 affected mRNA decay . In the present paper, we show that the missense mutation sup45-103 (L21S) alters degradation of PTC-containing mRNAs by NMD. However, we have previously shown that this mutation does not affect the eRF1-eRF3 interaction , indicating that this allele has an inhibitory effect on NMD that is independent on eRF1-eRF3 binding. This result demonstrates that eRF1 mutation affecting PTC-containing mRNA decay by NMD does not obligatory alters the eRF1-eRF3 interaction.
A role for the Upf1 protein, essential for NMD, in translation termination first became evident when a set of mutations were isolated in the UPF1 gene that separated the mRNA decay function from its activity in modulating premature termination [33, 34]. Subsequent studies have shown that deletion of either UPF2 or UPF3 can also lead to a nonsense suppression phenotype [7, 9, 11, 13, 35]. In addition, it was shown that upf1Δ mutation causes a general decrease in the efficiency of translation termination at UAG, UAA, and UGA stop codons .
In this work, we have shown that deletion of UPF1 does not affect ade1-14 mRNA level, but results in allosuppression of ade1-14 mutation in sup45 nonsense mutants therefore revealing that deletion of UPF1 has a synergistic effect with sup45-n mutants. Similar allosuppressor effect has also been shown for deletion of UPF1 in combination with [PSI+] [30, 36]. Based on this additive effect, Keeling et al.  proposed that upf1Δ mutation and [PSI+] influence the termination process in distinct ways. Our results suggest that this could be also the case for upf1Δ and sup45 mutations.
We found that deletion of the UPF1 gene affects several other sup45 phenotypes, such as temperature sensitivity, paromomycin sensitivity and viability of sup45 mutants. It is known that deletion of UPF1 gene in yeast does not cause any detectable phenotypic effects except respiratory deficiency  and nonsense suppression [7, 9, 13, 33–35]. Also telomere length is affected by deletions of UPF1-3 genes . How UPF1 deletion could affect sup45 phenotypes? We can not exclude an indirect effect of UPF1 deletion on sup45 phenotypes. It has been reported that NMD controls the mRNA levels of several hundred of wild-type genes [24, 26]. One can hypothesize that depletion of Upf1 could affect the expression of some translation apparatus components (e.g. tRNA genes) which themselves influence the viability of sup45 mutants. Indeed, the presence of SUQ5 mutation, a mutant suppressor tRNASer, increases the viability of sup45-n mutants . Alternatively, since inactivation of the NMD pathway by upf1Δ mutation does not increase the steady-state levels of wild-type and mutant SUP45 mRNAs and does not cause a change in the amount of eRF1 protein, we propose that the effect of NMD on sup45 phenotypes is probably via a change in the stoichiometry of factors involved in translation termination and NMD. In contrast to mammals, Upf proteins of S. cerevisiae are present at very low intracellular concentrations . Considering that in sup45-102 and sup45-107 nonsense mutants the amount of eRF1 was estimated as 8% and 17% of wild-type level, respectively , in mutant cells eRF1 and Upf1 proteins are probably present in stoichiometric amounts. Possibly, in wild-type cells, Upf1 is not preventing normal termination because its amount is ten times lower than eRF1, but in the case of their presence in stoichiometric amounts in sup45 nonsense mutants binding of Upf1 to eRF1 could result in a defective complex formation that blocks termination. This hypothesis is supported by finding that viability of sup45 nonsense mutants depends also on Upf2 or Upf3 proteins. There is a possibility that effect of Upf2 or Upf3 depletion is indirect and is under control of Upf1. It was shown that in mammalian cells a depletion of Upf2 or Upf3 reduces the amount of the phosphorylated form of Upf1 possibly preventing Upf1 dissociation from eRF3 and eRF1 . Phosphorylation of Upf1 and Upf2 was also shown in S. cerevisiae [41, 42], an indication that this mechanism might operate in yeast cells as well.
From recent studies, it appears more and more clear that translation termination and mRNA stability are intimately linked and our results demonstrate that eRF1 is also an essential factor linking these two processes.
In the present work, we have shown hat nonsense and missense mutations in SUP45 gene lead to stabilization of CYH2, a PTC-containing pre-mRNA degraded by NMD, and to accumulation of his7-1 mRNA. At the same time, sup45 mutations do not promote accumulation of other nonsense-containing transcripts, such as ade1-14 or lys9-A21, despite efficient suppression of these mutations. Thus sup45 mutations specifically affect PTC-containing mRNA subjected to NMD. Deletion of UPF1 results in allosuppression of ade1-14 mutation in sup45 nonsense mutants and leads to an increase in CYH2 pre-mRNA abundance therefore revealing that deletion of UPF1 has a synergistic effect with sup45-n mutants. This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.
Models explaining increased viability of sup45 nonsense mutants in the absence of Upf1, Upf2 or Upf3 proteins are proposed. First, the depletion of Upf1 could affect the expression of some translation apparatus components (e.g. tRNA genes) which themselves influence the viability of sup45 mutants. Second, a change in the stoichiometry of factors involved in translation termination and NMD provides the effect of NMD on sup45 phenotypes.
Yeast strains, plasmids and growth conditions
Strains used in this study
MATα ade1-14(UGA) his7-1(UAA) trp1-289(UAG) lys9-A21(UAA) ura3-52 leu2-3,112
MATa ade1-14(UGA) his7-1(UAA) trp1 ura3 upf1::kanMX4
MATa ade1-14 his7-1 lys2-87 met13-A1 thr4-B15 trp1 ura3-52 leu2-3,112 SUP35::TRP1
MATa his3Δ1 ura3Δ0 leu2Δ0 met15Δ0 upf1::kanMX4
MATa his3Δ1 ura3Δ0 leu2Δ0 met15Δ0 upf2::kanMX4
MATa his3Δ1 ura3Δ0 leu2Δ0 met15Δ0 upf3::kanMX4
MATa ade1-14 his3 leu2 lys2 ura3 sup45::HIS3 [pRS316/SUP45]
MATα ade1-14 his3 leu2 lys2 ura3 sup45::HIS3 [pRS316/SUP45] upf1::kanMX4
MATα ade1-14 his3 leu2 lys2 ura3 sup45::HIS3 [pRS316/SUP45]
MATa ade1-14 his3 leu2 lys2 met15Δ ura3 sup45::HIS3 [pRS316/SUP45] upf2::kanMX4
MATa ade1-14 his3 leu2 ura3 lys2 sup45::HIS3 [pRS316/SUP45]
MAT α ade1-14 his 3 leu2 ura3 lys2 sup45::HIS3 [pRS316/SUP45] upf3::kanMX4
MATα ade1-14 his3 lys2 ura3-52 leu2-3,112 trp1 sup45::HIS3 [pRS315/SUP45]
MATα ade1-14 his3 lys2 ura3-52 leu2-3,112 trp1 sup45::HIS3 [pRS315/SUP45] upf1::kanMX
The haploid SUP45::HIS3 [CEN URA3 SUP45] and SUP45::HIS3 UPF::kanMX4 [CEN URA3 SUP45] strains were used in "plasmid shuffle". These strains were transformed with [CEN LEU2 sup45] plasmids. Transformants, selected on -Ura-Leu medium, were velveteen replica plated onto 5-FOA medium, which counterselects against URA3 plasmids . Growth was also assayed using serial dilutions of overnight cultures with OD600 = 1. Serially (10-fold) diluted yeast cell cultures were spotted on plates containing 5-FOA to determine the ability of the sup45 mutant alleles to support cell growth in the presence and absence of any one of three UPF genes. The wild-type yeast SUP45 gene carried on the URA3 plasmid eliminates because 5-FOA is toxic to cells expressing the URA3 gene. The same serially diluted cultures were also spotted on plates lacking leucine and uracil to estimate the total number of cells analyzed.
Sequencing of the alleles his7-1, lys9-A21 and trp1-289
Yeast DNA was prepared using genomic DNA purification Kit (Promega). DNA fragments, corresponding to ORFs were amplified with the following primers:
HIS7 (202 GGCAGCTATTGAAGTAGCAGTATCCAG and
LYS9 (192 CAGCAATAGATGATAGAAAGTAGCACAG and
TRP1 (188 GAGGGAGGGCATTGGTGACTATTG and
For each allele at least two independent PCR-products were sequenced using the following primers:
HIS7 (195 GATGTACGTACTAATGACCAAGGTG and
LYS9 (197 GCTAAATACTGGAAAGACGGAAAG and
TRP1 (177 GTCTGTTATTAATTTCACAGGTAGTTC).
Analysis of mRNA steady-state levels
Total RNA was prepared by hot-phenol extraction method from yeast culture grown in YPD medium to log phase OD600 = 0.5-0.8 as described . Five micrograms of each RNA sample were separated on a 1.2% agarose gel, containing 3% formaldehyde and transferred to nylon membrane, Z-probe (Bio-Rad). SUP45, HIS7, CYH2 and ACT1 transcripts were detected using gene-specific 32P-radiolabelled DNA probes. Radioactive signals were directly detected and quantified by STORM Phosphor Imager system (Molecular dynamics, USA).
Probes were synthesized using following oligos:
82 CATTTCGGCTTGTCTCC and 83 TCTGGCATCTAGTGATTAAATTC (for SUP45), 195 GATGTACGTACTAATGACCAAGGTG and 196 GTTACTTCATCCGCACCCTGTTGG (for HIS7), 186 GACTAGAAAGCACAGAGGTCACGTC and 187 GACTAGAAAGCACAGAGGTCACGTC (for CYH2), 175 CGAAGACTGAACTGGACGGTATATTG and 176 CCCTGTCAATGTTTCATAAGCCTC for ADE1, 192 CAGCAATAGATGATAGAAAGTAGCACAG and 193 CAAGCTTCAGGAACTACACTCTC (for LYS9), 215 AGGCTGTAATGGCTTTCTGGTGGGATGGGA and 216 GATATGTGCTATCCCGGCCGCCTCCATCAC (for scR1).
Hind III-Xba I fragment of plasmid pSK/actin was used as a probe for ACT1 (M. Vedel, Institut of M. Curie, Paris).
Protein isolation, SDS-PAGE electrophoresis, and western blotting were performed as described previously . Antibodies specific to eRF1 and eRF3 were described previously [18, 20], monoclonal anti-α-tubulin antibodies were described before . eRF1 and eRF3 and α-tubulin signals were detected using alkaline-phosphatase-coupled anti-rabbit immunoglobulin G secondary antibodies (Jackson) (for eRF1 and eRF3) or alcaline-phosphatase-coupled anti-mouse immunoglobulin G secondary antibodies (Jackson) (for tubulin) by Amersham ECF system (Amersham Pharmacia Biotech). Signals were quantified with STORM 840 Phosphor-Imager (Molecular dynamics, USA) and ImageQuantNT 5.2 software.
This work was supported by grant from CRDF ST-012, S.C. is supported by grants from NATO and FEBS. We are very grateful to M. Culbertson and K. Volkov for kindly providing plasmid constructs, H.B.Osborne for critical reading of the manuscript, D. Kiktev, A. Petrova and J-P. Gagné for technical assistances. We also thank Michel Philippe for his support.
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