Anton A. Nizhnikov

[NSI+]: a novel non-Mendelian nonsense suppressor determinant in Saccharomyces cerevisiae

Non-Mendelian determinants that control heritable traits in yeast are subdivided into two major groups—one that includes DNA- or RNA-based elements and another that comprises protein-based factors that are analogous to mammalian prion. All yeast non-Mendelian determinants show dominant inheritance, and some of them demonstrate cytoplasmic infectivity. Only prions, however, harbor-specific features, such as high frequency of induction following overproduction of prion-encoding protein, loss of the protein’s normal function, and reversible curability. Here, we describe a novel nonchromosomal determinant that, in addition to [PSI+] and [ISP+], is involved in epigenetic control of nonsense suppression. This determinant, which we have designated [NSI+], causes nonsense suppression in the strains bearing the N-terminal-deleted or -modified SUP35 gene, but has no manifestation in the strains with the intact copy of SUP35. [NSI+] shows dominant non-Mendelian inheritance, reversible curability and may be transmitted by cytoduction, albeit with low frequency. Similar to yeast prions, this determinant can be cured by deletion or mutational inactivation of Hsp104. We have shown that [NSI+] does not correspond to the already identified yeast prions. Based on the data obtained, we hypothesize that [NSI+] is a novel prion factor involved in epigenetic control of nonsense suppression.

Proteomic Screening for Amyloid Proteins

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

Amyloids: from pathogenesis to function

The term “amyloids” refers to fibrillar protein aggregates with cross-ß structure. They have been a subject of intense scrutiny since the middle of the previous century. First, this interest is due to association of amyloids with dozens of incurable human diseases called amyloidoses, which affect hundreds of millions of people. However, during the last decade the paradigm of amyloids as pathogens has changed due to an increase in understanding of their role as a specific variant of quaternary protein structure essential for the living cell. Thus, functional amyloids are found in all domains of the living world, and they fulfill a variety of roles ranging from biofilm formation in bacteria to long-term memory regulation in higher eukaryotes. Prions, which are proteins capable of existing under the same conditions in two or more conformations at least one of which having infective properties, also typically have amyloid features. There are weighty reasons to believe that the currently known amyloids are only a minority of their real number. This review provides a retrospective analysis of stages in the development of amyloid biology that during the last decade resulted, on one hand, in reinterpretation of the biological role of amyloids, and on the other hand, in the development of systems biology of amyloids, or amyloidomics.

[NSI+] determinant has a pleiotropic phenotypic manifestation that is modulated by SUP35, SUP45, and VTS1 genes

We recently discovered the novel non-chromosomal determinant in Saccharomyces cerevisiae [NSI+] (nonsense suppression inducer), which causes omnipotent nonsense suppression in strains where the Sup35 N-terminal domain is deleted. [NSI+] possesses yeast prion features and does not correspond to previously identified yeast prion determinants. Here, we show that [NSI+] enhances nonsense codon read-through and inhibits vegetative growth in S. cerevisiae. Using a large-scale overexpression screen to identify genes that impact the phenotypic effects of [NSI+], we found that the SUP35 and SUP45 genes encoding the translation termination factors eRF3 and eRF1, respectively, modulate nonsense suppression in [NSI+] strains. The VTS1 gene encodes an NQ-enriched RNA-binding protein that enhances nonsense suppression in [NSI+] and [nsi−] strains. We demonstrate that VTS1 overexpression, like [NSI+] induction, causes translational read-through and growth defects in S. cerevisiae.

Identification of genes encoding potentially amyloidogenic proteins that take part in the regulation of nonsense suppression in yeast Saccharomyces cerevisiae

Previously, we demonstrated that SUP35 N-terminal deletion creates a specific genetic back-ground permitting the identification of novel genes and epigenetic determinants controlling nonsense suppression. In the present study, using a genomic screen, we found three genes encoding potentially amyloidogenic proteins, whose overexpression affects nonsense suppression in the strain producing chimeric Aβ-Sup35MC protein on the background of SUP35 deletion encoding releasing factor eRF3. These genes, NAB2, NAB3, and VTS1, were found to participate in the regulation of nonsense suppression in yeast S. cerevisiae.

Yeast chaperone Hsp104 controls gene expression at the posttranscriptional level

Yeast chaperone Hsp104 is known as a protein responsible for dissociation of aggregates of heat-damaged proteins and prion aggregates into smaller pieces or monomers. The effects of Hsp104 on PrP-GFP and GFP were analyzed. PrP-GFP forms high-molecular-weight aggregates, whereas GFP is unable to aggregate in yeast cells. Hsp104 proved to regulate the amount of PrP-GFP and GFP in yeast cells, and the direction of chaperone action depended on the promoters controlling the production of these proteins. Overproduction of Hsp104 increased the levels of PrP-GFP and GFP when their genes were controlled by the CUP1 promoter. In contrast, overproduction of Hsp104 decreased the levels of PrP-GFP and GFP in the case of their expression under the control of the GPD promoter. The effects of Hsp104 were not related to any changes in the contents of mRNAs of the genes under investigation nor to the ability of the proteins to form aggregates. Thus, the Hsp104 functions were not confined to dissociation of protein aggregates. Hsp104 was assumed to regulate gene expression at the posttranscriptional level.

Interactions of [ NSI + ] prion-like determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae

Previously we characterized [NSI+], determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI+]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI+]. We demonstrated nonsense suppression in the [NSI+] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI+] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI+] strains and narrow the range of candidates for [NSI+] determinant.

Prions, amyloids, and RNA: Pieces of a puzzle.

ABSTRACT Amyloids are protein aggregates consisting of fibrils rich in β-sheets. Growth of amyloid fibrils occurs by the addition of protein molecules to the tip of an aggregate with a concurrent change of a conformation. Thus, amyloids are self-propagating protein conformations. In certain cases these conformations are transmissible / infectious; they are known as prions. Initially, amyloids were discovered as pathological extracellular deposits occurring in different tissues and organs. To date, amyloids and prions have been associated with over 30 incurable diseases in humans and animals. However, a number of recent studies demonstrate that amyloids are also functionally involved in a variety of biological processes, from biofilm formation by bacteria, to long-term memory in animals. Interestingly, amyloid-forming proteins are highly overrepresented among cellular factors engaged in all stages of mRNA life cycle: from transcription and translation, to storage and degradation. Here we review rapidly accumulating data on functional and pathogenic amyloids associated with mRNA processing, and discuss possible significance of prion and amyloid networks in the modulation of key cellular functions.

Modulation of efficiency of translation termination in Saccharomyces cerevisiae

Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy.

SARP: A Novel Algorithm to Assess Compositional Biases in Protein Sequences

The composition of a defined set of subunits (nucleotides, amino acids) is one of the key features of biological sequences. Compositional biases are local shifts in amino acid or nucleotide frequencies that can occur as an adaptation of an organism to an extreme ecological niche, or as the signature of a specific function or localization of the corresponding protein. The calculation of probability is a method for annotating compositional bias and providing accurate detection of biased subsequences. Here, we present a Sequence Analysis based on the Ranking of Probabilities (SARP), a novel algorithm for the annotation of compositional biases based on ranking subsequences by their probabilities. SARP provides the same accuracy as the previously published Lower Probability Subsequences (LPS) algorithm but performs at an approximately 230-fold faster rate. It can be recommended for use when working with large datasets to reduce the time and resources required.