Using recombinant eRF3 and cellular extracts, we have shown that yeast eRF3 and Pab1p can interact. That interaction was demonstrated by the two-hybrid approach to be direct. As shown previously for mammalian eRF3 and PABP (46
), we found that yeast eRF3-Pab1p interactions are mediated by the very weakly conserved N-terminal domain of eRF3. At least in yeast, eRF3 plays an essential role in translation termination, interacting with eRF1 via the C-terminal GTPase domain (29
). Both factors are said to be essential for translation termination in yeast (90
). As shown in Fig. , eRF3 also contains domains within its sequence that interact with Upf1, Upf2, and Upf3 proteins, which are the components of the surveillance complex (99
), and with Sla1, a protein involved in the assembly of the cortical actin cytoskeleton (4
). Although genetic data suggest a functional relationship between eRF3 and Hsp104 (14
), a physical interaction between these proteins could not be demonstrated (84
). Upf1-like helicase Mtt1 also interacts with eRF3, but the domain of interaction within eRF3 was not precisely localized; two interaction domains were described previously (20
Our data suggest that the domain of interaction of eRF3 with Pab1p is situated between those interacting with Sla1 and Upf1 (Fig. ).
The Pab1p-interacting region in eRF3.
Previously yeast eRF3 was schematically divided into three domains: the N-terminal prion-determining domain (PrD, aa 1 to 123), the middle domain of unknown function (M, aa 124 to 253), and the C-terminal domain, structurally similar to translation elongation factor eEF1A and essential for cell viability (C, aa 254 to 685) (96
). Further studies showed that overexpression of only the first 113 aa of eRF3 was sufficient for [PSI+
] establishment (55
) and that an N-terminal polypeptide consisting of residues 2 to 114 spontaneously aggregated in vitro into amyloid-like filaments (54
). Recently, the minimum length of PrD was defined as aa 1 to 97 (76
aa 1 to 113 of eRF3 are sufficient to mediate the interaction with Pab1p, suggesting that the Pab1p-interacting domain could overlap with PrD. However, several lines of evidence indicate that Pab1p does not influence [PSI+
] propagation. Firstly, [PSI+
]-no-more mutations do not affect eRF3-Pab1p two-hybrid interaction. Secondly, overexpression of PAB1
does not cure the [PSI+
] phenotype or solubilize detectable amounts of eRF3. Finally, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI+
), were not modified by excess Pab1p, in contrast to what was obtained with Sla1p. These data also suggest that the eRF3 domains involved in Pab1p and Sla1 interactions are different.
Similar to Pab1p, eRF1, another eRF3-interacting protein, does not influence [PSI+
] propagation (23
). Possibly, the [PSI+
] complex, once formed, is resistant to the putative influence of eRF3-interacting proteins. The only protein influencing [PSI+
] propagation identified so far is Hsp104. However, the mechanism of its action remains unclear because no direct interaction with eRF3 could be shown (see reference 85
for a review).
An interaction between Pab1p and the yeast Hsp70 homologue Ssa1 was recently shown (45
). Interestingly an excess of Ssa1 could protect [PSI+
] from curing by overexpression of Hsp104 (71
); this fact suggests that Ssa1 and eRF3 could form a transient complex. Depletion of Ssa1 affects translation initiation, reducing Pab1p-eIF4G interaction (45
). Possibly, Ssa1 modulates [PSI+
] curing or translation initiation through Pab1p, but this interaction remains to be shown.
Our data show that the interaction of eRF3 with Pab1p is evolutionarily conserved. In particular we have shown that human eRF3 could interact with yeast Pab1p via the same N-terminal domain as with human PABP. However, sequence analysis failed to reveal any significant homology of the primary structures of the identified domains of S. cerevisiae
eRF3 and human eRF3. Secondary structure predictions using the SOPM algorithm (37
) indicated mostly random coil conformation for PABP-interacting regions of all eRF3 homologues (data not shown). Possibly this region could form a helical structure only when bound to Pab1p-C. This has been shown previously to occur for Paip2 when bound to PABP-C (57
). Interestingly the predicted PABC-interacting consensus sequence (LNVNAKPFVP) of Paip2 has also been found in eRF3 proteins from different species (57
). In all eRF3 proteins analyzed (hGSPT1, mGSPT2, xSUP35, and yeast eRF3) the PABP-interacting sequence is located near a short helical region (Fig. ). A comparative secondary structure analysis reveals aa 132 to 140 of yeast eRF3 (qqkqAaPkpk) as a potential Pab1p-interacting region (Fig. ).
FIG. 7. Secondary structure prediction for the PABP-interacting region in various eRF3 homologues. Alignment of the N-terminal parts of Xenopus (xeRF3, aa 51 to 87), human (hGSPT1, aa 48 to 85), mouse (mGSPT2, aa 35 to 76), and yeast (yeRF3, aa 109 to 149) eRF3 (more ...) A role of PABP in translation termination.
Especially for S. cerevisiae
, many data had been accumulated on the PABP, which was initially characterized through its ability to strongly bind to poly(A). Clearly Pab1p plays essential roles in mRNA metabolism and translation. More specifically, in yeast, the protein regulates the poly(A) tail length during the polyadenylation reaction (2
). It inhibits the activity of the purified poly(A) polymerase (64
) and is required for Pab1p-dependent poly(A) nuclease activity to control mature mRNA tail length (11
). Pab1p is able to prevent mRNA decay independently of the presence of a poly(A) tail if tethered to the mRNA (16
). Furthermore, PABP prevents access of a 3′-to-5′ poly(A)-specific exoribonuclease activity in mammalian cells (6
). It is known that Pab1p participates in translation initiation via its interaction with eIF4G (91
). More recently it was shown that the interaction with eIF4G, which also occurs in higher eukaryotes (48
), serves to integrate the functions of the 5′ cap and the 3′ poly(A) tail in translation initiation (41
). Pab1p also stimulates poly(A)-dependent and cap-dependent translation by different mechanisms in vitro (73
). All these activities involve the N-terminal part of Pab1p, which is essential and consists of four RRMs.
In this work, we demonstrate that Pab1p has an antisuppressor effect in vivo. Moreover we show that this effect requires the site of the binding of Pab1p to eRF3, suggesting that Pab1p has an antisuppressor effect on translation termination through its interaction with eRF3. This is a completely new function for Pab1p. Such a role for PABP in higher eukaryotes remains to be demonstrated, but the fact that human PABP and eRF3 also interact suggests that this could be the case.
Nonsense suppression occurs when a near-cognate tRNA successfully competes with the termination factors at a nonsense codon; amino acid incorporation into the peptide chain instead of translation termination occurs at the site of nonsense mutation. Mutations that result in the nonsense suppression phenotype have been identified in several genes. Among them were the SUP45
genes, encoding peptidyl release factors eRF1 and eRF3, and also UPF1
, whose disruption promotes suppression of certain nonsense alleles (61
). Recently another gene, MTT1
, modulating the efficiency of translation termination and interacting with eRF3, was described (20
Drugs that specifically alter translation termination are not known. For this reason, we looked for different situations where translation termination activity was weak, in order to study the functional significance of Pab1p and eRF3 interaction in this essential step of gene expression.
Paromomycin is an aminoglycoside antibiotic influencing translation fidelity. Recent data clearly show that it interacts with a highly conserved region in the 3′ end of 16S rRNA and that its binding induces a local conformational change in this decoding region (33
). Also it has been shown previously that the structures of prokaryotic and eukaryotic decoding region A sites are similar (66
) in most details and that paromomycin binds to this decoding site in both prokaryotic and eukaryotic ribosomes. Thus, such binding is highly specific and so paromomycin has a direct influence on translation termination.
It is known that mutations in genes SUP35
cause paromomycin sensitivity (for a review see reference 49
). Moreover, a nonsense mutation in yeast can be phenotypically suppressed with paromomycin (74
), which also increases the efficiency of [PSI+
]-dependent suppression (75
). It was found previously that a mutation in a gene encoding a translation initiation factor could also cause paromomycin sensitivity, and it was proposed elsewhere that paromomycin could affect the function of any protein involved in the decoding process (40
Our data show that PAB1 overexpression in a SUP35 mutant could restore paromomycin resistance and that this effect depends on the presence of the Pab1p C terminus, which is necessary for the interaction with eRF3. With a vector-based assay system we measured the termination activity and confirmed that the overexpressed Pab1p could compensate for the deleterious effects of the eRF3 mutation on translation termination machinery. Quantitative mRNA analysis has not revealed destabilization of nonsense mRNA by Pab1p overexpression. Thus, the implication of our results is that Pab1p overexpression has a an antisuppressor effect which is obtained via its interaction with eRF3 on the termination machinery.
These data are in agreement with the effect of overexpressed Pab1p on [PSI+
]-dependent suppression. In [PSI+
] cells, most of the eRF3 (Sup35) is converted from a soluble, active state into an insoluble, inactive state that enhances the suppression of nonsense mutations (see reference 85
for a review).
We have shown that together the presence of paromomycin and low temperature (18°C) are lethal for [PSI+] cells. Overexpression of full-length Pab1p but not that of its C-terminally truncated variant restored viability in these conditions. Again, the eRF3 binding domain of Pab1p is necessary, suggesting that overexpressed Pab1p could restore termination of translation via eRF3 interaction. We failed to detect any effect of Pab1p on [PSI+] propagation or eRF3 aggregation, although we could not exclude the possibility that even a limited solubilization of eRF3 could be sufficient to restore termination (see below). All these findings together suggest that overexpressed Pab1p could restore termination of translation via eRF3 interaction.
Overexpression of Pab1p leads to an antisuppressor effect for all stop codons in the [PSI+
] strain. Also, it decreases the phenotypic suppression of the his7-1
(UAA) nonsense mutation caused by paromomycin in a wild-type strain. The inability of overexpressed PAB1
to act against suppression of the UAA codon in the sup35-21
strain could be connected with the nature of the sup35-21
mutation, which changes a glutamine codon to a UAA stop codon at aa 422. It has been shown previously that aa 254 to 685 of eRF3 are essential for cell viability and that C-terminally truncated eRF3 (aa 1 to 482) is unable to support viability (97
), and so the sup35-21
strain should be viable only in the case of readthrough of the UAA codon; this has likewise been proposed for the sup35-2
(UAG) mutation (108
). Indeed, Western blot analysis has shown that the expression of full-length eRF3 in the sup35-21
strain was lower than that in the wild-type strain. Viable but thermosensitive nonsense mutations have been isolated in several essential genes of S. cerevisiae
) and of Salmonella enterica
serovar Typhimurium (52
). In the case of the supK584
mutation, which caused an opal (UGA) substitution in the prfB
gene of S. enterica
serovar Typhimurium, encoding RF2 release factor, it has been shown previously that this mutation reduces the cellular amount of RF2 (52
). Such a reduction in RF2 level causes inefficient termination of translation and leads to autosuppression. An analogous mechanism has also been proposed for the sup35-2
). Overexpression of PAB1
only slightly decreases the level of eRF3, suggesting that the antisuppressor effect of Pab1p is not mediated by regulating the level of eRF3.
We hypothesize that the mutation CAA (422Gln)→UAA leads to a misincorporation of the amino acid during the readthrough reaction. This change of amino acid would alter the structure of eRF3, leading to the suppression phenotype and thermosensitivity. PAB1 overexpression leads to reversal of this phenotype by increasing termination efficiency. It is unlikely that Pab1p bypasses the eRF3 function in termination, as the overexpression effect requires the eRF3 binding domain. There are several possible ways in which Pab1p overexpression leads to an antisuppressor phenotype.
The first possibility is that eRF3 cannot be recruited to form an active complex with eRF1 because of a conformation problem in the sup35-21 strain and because of aggregation in [PSI+] cells. Pab1p possibly helps to recruit eRF3 and/or restores functional conformation of the altered eRF3 protein during the termination reaction in the ribosome. Results of [PSI+] strain analysis showed that overexpressed Pab1p is unable to solubilize detectable levels of eRF3 protein, although it cannot be excluded that a restricted amount of eRF3 directed to the translation machinery could have an antisuppressor effect. This implies that Pab1p interacts with eRF3 during the termination reaction and promotes its activity.
Another possibility is based on the fact that release factors need to be recycled: the number of eRF1 and eRF3 termination factors is limited compared to that of ribosomes (about one copy of eRF3 per 20 ribosomes [26
]). Termination (release of nascent protein from the ribosome) but also posttermination events (release and/or regeneration of the active form of ribosome and associated factors and possibly reinitiation) may determine the kinetics of the termination reaction in a cellular context. Inhibition of recycling may result in reduced translation termination efficiency. During the translation termination a surveillance complex is assembled and searches 3′ of the termination codon for specific signals that target the mRNA for rapid degradation. A dynamic model for the surveillance complex assembly was proposed according to which dissociation of eRF1 allows either Upf2 or Upf3 to bind to the eRF3-Upf1 complex (99
). Since eRF3 blocks the ATPase-helicase activity of Upf1p (22
), dissociation of eRF3 from the Upf complex—but not necessarily from the ribosome--would be necessary to form the active surveillance complex. In contrast to the general deadenylation-dependent pathway, degradation by the NMD pathway is independent of prior deadenylation of the mRNA and Pab1p was shown previously to be unable to prevent the decay of mRNA subjected to NMD (16
). Consequently, it is not likely that Pab1p outcompetes Upf proteins for binding to eRF3, because that would prevent NMD. We do not know the fate of the translation machinery components after peptide release, but eRF3 has previously been found associated with ribosomes and 40S subunits (26
), suggesting that eRF3 remains associated with the 40S subunit after termination. As the function of the Pab1p-poly(A) tail complex is to recruit the 40S subunit on the mRNA in order to stimulate its translation (92
), we hypothesize that eRF3 could mark a ribosome that has already finished one round of translation and that would be reloaded on the mRNA in order to ensure efficiency of translation. According to this model, Pab1p overexpression would act to stimulate eRF3 in the posttermination recycling step, allowing efficient termination in the next rounds of translation of the same or other mRNAs. This model postulates that mutants in which posttermination events do not occur properly could also be detected by a nonsense suppression phenotype, as observed for Upf-deletion strains. Development of in vitro systems that uncouple posttermination events from peptide release will be needed to demonstrate the role of all these factors. This work will be of major interest, as the 5′ and 3′ ends of the mRNA may interact to form a closed-loop structure.