S. E. Moskalenko

Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae

BackgroundTermination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.ResultsWe have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.ConclusionsNonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Mouse GSPT2, but not GSPT1, can substitute for yeast eRF3 in vivo

Background The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2.

Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae

BackgroundThe 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.ResultsIn 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.ConclusionThis is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.

Characterization of Missense Mutations in the SUP45 Gene of Saccharomyces cerevisiae Encoding Translation Termination Factor eRF1

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1–eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.

Increased tRNA level in yeast cells with mutant translation termination factors eRF1 and eRF3

We have earlier characterized Saccharomyces cerevisiae strains with mutations of essential SUP45 and SUP35, which code for translation termination factors eRF1 and eRF3, respectively. In this work, the sup45 and sup35 nonsense mutants were compared with respect to the levels of eight tRNAs: tRNATyr, tRNAGln, tRNATrp, tRNALeu, tRNAArg (described as potential suppressor tRNAs), tRNAPro, tRNAHis, and tRNAGly. The mutants did not display a selective increase in tRNAs, capable of a noncanonical read-through at stop codons. Most of the mutations increased the level of all tRNAs under study. The mechanisms providing for the viability of the sup45 and sup35 nonsense mutants are discussed.

To CURe or not to CURe? Differential effects of the chaperone sorting factor Cur1 on yeast prions are mediated by the chaperone Sis1

Yeast self‐perpetuating protein aggregates (prions) provide a convenient model for studying various components of the cellular protein quality control system. Molecular chaperones and chaperone‐sorting factors, such as yeast Cur1 protein, play key role in proteostasis via tight control of partitioning and recycling of misfolded proteins. In this study, we show that, despite the previously described ability of Cur1 to antagonize the yeast prion [URE3], it enhances propagation and phenotypic manifestation of another prion, [PSI+]. We demonstrate that both curing of [URE3] and enhancement of [PSI+] in the presence of excess Cur1 are counteracted by the cochaperone Hsp40‐Sis1 in a dosage‐dependent manner, and show that the effect of Cur1 on prions parallels effects of the attachment of nuclear localization signal to Sis1, indicating that Cur1 acts on prions via its previously reported ability to relocalize Sis1 from the cytoplasm to nucleus. This shows that the direction in which Cur1 influences a prion depends on how this specific prion responds to relocalization of Sis1.

SFP1‐mediated prion‐dependent lethality is caused by increased Sup35 aggregation and alleviated by Sis1

[PSI+] is the prion form of the translation termination factor Sup35 (eRF3); [PSI+] strains display nonsense suppression. Another prion‐like element, [ISP+], is linked to antisuppression in a specific background. Transcriptional regulator Sfp1 was shown to be responsible for [ISP+] propagation. In this work, we identified SFP1 as a multicopy inducer of [PSI+]‐dependent lethality. Sfp1 is likely to up‐regulate transcription of genes encoding release factors; however, its overproduction increases Sup35, but not Sup45 protein level. Using the synthetic lethality test, we compared the effects of SFP1 and SUP35 over‐expression on the viability of [PSI+] strains. Together with an observation that Sfp1 overproduction leads to an increased accumulation of Sup35 in [PSI+] aggregates, we suggest that excess Sfp1 causes [PSI+] toxicity. Even though SUP45 over‐expression is known to compensate for the [PSI+]‐dependent lethality, it fails to do so when the lethality is caused by SFP1 over‐expression. We discovered that the increased levels of Hsp40 chaperone Sis1 alleviate prion toxicity caused by either SFP1 or SUP35 over‐expression and revert back to normal distribution of Sup35 between monomers and aggregate fractions. Finally, we showed that Sfp1 partially colocalizes with Sup35 aggregates, which may contribute to another mechanism of Sfp1‐derived [PSI+] prion toxicity.

The translation termination factor eRF1 (Sup45p) of Saccharomyces cerevisiae is required for pseudohyphal growth and invasion

Mutations in the essential genes SUP45 and SUP35, encoding yeast translation termination factors eRF1 and eRF3, respectively, lead to a wide range of phenotypes and affect various cell processes. In this work, we show that nonsense and missense mutations in the SUP45, but not the SUP35, gene abolish diploid pseudohyphal and haploid invasive growth. Missense mutations that change phosphorylation sites of Sup45 protein do not affect the ability of yeast strains to form pseudohyphae. Deletion of the C-terminal part of eRF1 did not lead to impairment of filamentation. We show a correlation between the filamentation defect and the budding pattern in sup45 strains. Inhibition of translation with specific antibiotics causes a significant reduction in pseudohyphal growth in the wild-type strain, suggesting a strong correlation between translation and the ability for filamentous growth. Partial restoration of pseudohyphal growth by addition of exogenous cAMP assumes that sup45 mutants are defective in the cAMP-dependent pathway that control filament formation.

Overexpression of genes encoding tRNATyr and tRNAGln increases the viability of Saccharomyces cerevisiae strains with nonsense mutations in the SUP45 gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNATyr and four genes encoding wild-type tRNAGln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNATyr and tRNAGln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.

Viable nonsense mutants for the SUP45 gene in the yeast Saccharomyces cerevisiae are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination factor eRF1 in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a trucated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37°C. When grown under nonpermissive conditions (37°C), cells of nonsense mutants sup45-104, sup45-105, and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.