Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation.

In yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles (sup35-n and sup45-n), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the sup35-218 nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (APC9, CDC23) and their activator CDC20, and increases in the expression of the transcription factor FKH1, the main cell cycle kinase CDC28, and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant sup35-n allele.

NOS1AP Interacts with α-Synuclein and Aggregates in Yeast and Mammalian Cells.

The NOS1AP gene encodes a cytosolic protein that binds to the signaling cascade component neuronal nitric oxide synthase (nNOS). It is associated with many different disorders, such as schizophrenia, post-traumatic stress disorder, autism, cardiovascular disorders, and breast cancer. The NOS1AP (also known as CAPON) protein mediates signaling within a complex which includes the NMDA receptor, PSD-95, and nNOS. This adapter protein is involved in neuronal nitric oxide (NO) synthesis regulation via its association with nNOS (NOS1). Our bioinformatics analysis revealed NOS1AP as an aggregation-prone protein, interacting with α-synuclein. Further investigation showed that NOS1AP forms detergent-resistant non-amyloid aggregates when overproduced. Overexpression of NOS1AP was found in rat models for nervous system injury as well as in schizophrenia patients. Thus, we can assume for the first time that the molecular mechanisms underlying these disorders include misfolding and aggregation of NOS1AP. We show that NOS1AP interacts with α-synuclein, allowing us to suggest that this protein may be implicated in the development of synucleinopathies and that its aggregation may explain the relationship between Parkinson's disease and schizophrenia.

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.

Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Gene Amplification as a Mechanism of Yeast Adaptation to Nonsense Mutations in Release Factor Genes.

Protein synthesis (translation) is one of the fundamental processes occurring in the cells of living organisms. Translation can be divided into three key steps: initiation, elongation, and termination. In the yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 and eRF3. These factors are encoded by the SUP45 and SUP35 genes, which are essential; deletion of any of them leads to the death of yeast cells. However, viable strains with nonsense mutations in both the SUP35 and SUP45 genes were previously obtained in several groups. The survival of such mutants clearly involves feedback control of premature stop codon readthrough; however, the exact molecular basis of such feedback control remain unclear. To investigate the genetic factors supporting the viability of these SUP35 and SUP45 nonsense mutants, we performed whole-genome sequencing of strains carrying mutant sup35-n and sup45-n alleles; while no common SNPs or indels were found in these genomes, we discovered a systematic increase in the copy number of the plasmids carrying mutant sup35-n and sup45-n alleles. We used the qPCR method which confirmed the differences in the relative number of SUP35 and SUP45 gene copies between strains carrying wild-type or mutant alleles of SUP35 and SUP45 genes. Moreover, we compare the number of copies of the SUP35 and SUP45 genes in strains carrying different nonsense mutant variants of these genes as a single chromosomal copy. qPCR results indicate that the number of mutant gene copies is increased compared to the wild-type control. In case of several sup45-n alleles, this was due to a disomy of the entire chromosome II, while for the sup35-218 mutation we observed a local duplication of a segment of chromosome IV containing the SUP35 gene. Taken together, our results indicate that gene amplification is a common mechanism of adaptation to nonsense mutations in release factor genes in yeast.

The Human NUP58 Nucleoporin Can Form Amyloids In Vitro and In Vivo.

Amyloids are fibrillar protein aggregates with a cross-ß structure and unusual features, including high resistance to detergent or protease treatment. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Several examples of nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human NUP153) are supposed to form amyloid fibrils. In this study, we demonstrated an ability of the human NUP58 nucleoporin to form amyloid aggregates in vivo and in vitro. Moreover, we found two forms of NUP58 aggregates: oligomers and polymers stabilized by disulfide bonds. Bioinformatic analysis revealed that all known orthologs of this protein are potential amyloids which possess several regions with conserved ability to aggregation. The biological role of nucleoporin amyloid formation is debatable. We suggest that it is a rather abnormal process, which is characteristic for many proteins implicated in phase separation.

Chromosome-level genome assembly and structural variant analysis of two laboratory yeast strains from the Peterhof Genetic Collection lineage.

Thousands of yeast genomes have been sequenced with both traditional and long-read technologies, and multiple observations about modes of genome evolution for both wild and laboratory strains have been drawn from these sequences. In our study, we applied Oxford Nanopore and Illumina technologies to assemble complete genomes of two widely used members of a distinct laboratory yeast lineage, the Peterhof Genetic Collection (PGC), and investigate the structural features of these genomes including transposable element content, copy number alterations, and structural rearrangements. We identified numerous notable structural differences between genomes of PGC strains and the reference S288C strain. We discovered a substantial enrichment of mid-length insertions and deletions within repetitive coding sequences, such as in the SCH9 gene or the NUP100 gene, with possible impact of these variants on protein amyloidogenicity. High contiguity of the final assemblies allowed us to trace back the history of reciprocal unbalanced translocations between chromosomes I, VIII, IX, XI, and XVI of the PGC strains. We show that formation of hybrid alleles of the FLO genes during such chromosomal rearrangements is likely responsible for the lack of invasive growth of yeast strains. Taken together, our results highlight important features of laboratory yeast strain evolution using the power of long-read sequencing.

Whole genome sequencing data and analyses of the underlying SUP35 transcriptional regulation for a Saccharomyces cerevisiae nonsense suppressor mutant.

Termination of translation in eukaryotes is governed by two release factors encoded by the SUP45 and SUP35 genes in Saccharomyces cerevisiae. Previously, a set of mutations in these genes had been obtained. However, the exact sequence change associated with one mutation, sup35-222, was not identified by Sanger sequencing of the SUP35 region. Presented here are whole-genome sequencing data for the sup35-222 strain, data on copy number variation in its genome along with supporting pulse-field gel electrophoresis experiment data, and the list of single-nucleotide variations that differentiate this strain and its wild-type ancestor. One substitution upstream the SUP35 gene was located in a sequence corresponding to the Abf1-binding site. Data obtained from the introduction of this variation from sup35-222 strain into a different wild-type strain, specifically, detection of a nonsense-suppressor phenotype accompanied by a decrease in the Sup35 protein level, are also presented in this article.

Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae.

BACKGROUND: Termination 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. RESULTS: We 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 degrees 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. CONCLUSIONS: Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.