Cross-talk between RNA and prions

As concepts evolve in mammalian and yeast prion biology, rather preliminary research investigating the interplay between prion and RNA processes are gaining momentum. The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. This "nonsense suppression" activity is investigated for its possible physiological role to engender on Saccharomyces cerevisiae the ability to respond to stress or variable growth conditions and thereby act as a capacitor to evolve. The interaction between prion and RNA is a two way street--the cell may have adopted RNA processes in translation to govern the presence of prions and the [PSI+] prion's nonsense suppressor phenotype may exhibit different growth phenotypes by its control of translation termination. RNA processes in the mammalian cell also effect and are affected by prions.     

Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion "strains," [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.     

Recent advances of research on the [PSI+] prion in Saccharomyces cerevisiae

Prion diseases such as bovine spongiform encephalopathy or Creutzfeldt-Jakob disease have been extensively studied in recent years. Research in this field is being done in highly secured laboratories because of potential transmission of prions to humans. Emerging similarities between mammalian and yeast prions allow using yeast-based assays to examine the activity of anti-prion drugs. Besides the intensively studied clinical aspects of prion diseases, the evolutionary aspects of prion proteins present in the yeast Saccharomyces cerevisiae are also extensively investigated. One of the key feature of prions, the ability to be stable in two alternative conformations, seems to play an important role in the evolution of this fungi, although some authors point out the negative influence of these particles upon yeast physiology. In this review, the most intensively studied fields of the research carried out on [PSI+] prion in yeast are summarized.     

The Genetic Control of the Formation and Propagation of the [PSI+] Prion of Yeast

It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI+] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learned much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI+] prion.     

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.     

A yeast-based assay to isolate drugs active against mammalian prions

Recently, we have developed a yeast-based (Saccharomyces cerevisiae) assay to isolate drugs active against mammalian prions. The initial assumption was that mechanisms controlling prion appearance and/or propagation could be conserved from yeast to human, as it is the case for most of the major cell biology regulatory mechanisms. Indeed, the vast majority of drugs we isolated as active against both [PSI(+)] and [URE3] budding yeast prions turned out to be also active against mammalian prion in three different mammalian cell-based assays. These results strongly argue in favor of common prion controlling mechanisms conserved in eukaryotes, thus validating our yeast-based assay and also the use of budding yeast to identify antiprion compounds and to study the prion world.     

Newly identified prions in budding yeast, and their possible functions

Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we summarize the characteristics of each prion element, and discuss their potential functional roles in yeast biology.     

Destabilizing interactions among [PSI(+)] and [PIN(+)] yeast prion variants

The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi-] or [pin-], respectively, or the multiple infectious amyloid-like forms called [PSI+] or [PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.     

PSI+] Prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies 'species barriers'

[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.     

The [PSI+] prion of yeast: a problem of inheritance

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.     

Yeast Prions

Prions (infectious proteins) analogous to the scrapie agent have been identified in Saccharomyces cerevisiae and Podospora anserina based on their special genetic characteristics. Each is a protein acting as a gene, much like nucleic acids have been shown to act as enzymes. The [URE3], [PSI+], [PIN+] and [Het-s] prions are self-propagating amyloids of Ure2p, Sup35p, Rnq1p and the HET-s protein, respectively. The [b] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI+] are clearly diseases, while [Het-s] and [b] carry out normal cell functions. Surprisingly, the prion domains of Ure2p and Sup35p can be randomized without loss of ability to become a prion. Thus amino acid content and not sequence determine these prions. Shuffleability also suggests amyloids with a parallel in-register b-sheet structure.     

Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures

Most prions (infectious proteins) are self-propagating amyloids (filamentous protein multimers), and have been found in both mammals and fungal species. The prions [URE3] and [PSI+] of yeast are disease agents of Saccharomyces cerevisiae while [Het-s] of Podospora anserina may serve a normal cellular function. The parallel in-register beta-sheet structure shown by prion amyloids makes possible a templating action at the end of filaments which explains the faithful transmission of variant differences in these molecules. This property of self-reproduction, in turn, allows these proteins to act as de facto genes, encoding heritable information.     

Prions

Prions were originally defined as infectious agents of protein nature, which caused neurodegenerative diseases in animals and humans. The prion concept implies that the infectious agent is a protein in special conformation that can be transmitted to the normal molecules of the same protein through protein-protein interactions. Until the 1990s, the prion phenomenon was associated with the single protein named PrP. Discovery of prions in lower eukaryotes, the yeast Saccharomyces cerevisiae and fungus Podospora anserina, suggests that prions have wider significance. Prions of lower eukaryotes are not related to diseases; their propagation caused by aggregation of prion-like proteins underlies the inheritance of phenotypic traits and most likely has adaptive significance. This review covers prions of mammals and lower eukaryotes, mechanisms of their appearance de novo and maintenance, structure of prion particles, and prospects for the treatment of prion diseases. Recent data concerning the search for new prion-like proteins is included. The paper focuses on the [PSI+] prion of S. cerevisiae, since at present it is the most investigated one. The biological significance of prions is discussed.     

Prion diseases of yeast: amyloid structure and biology

Prion "variants" or "strains" are prions with the identical protein sequence, but different characteristics of the prion infection: e.g. different incubation periods for scrapie strains or different phenotype intensities for yeast prion variants. We have shown that infectious amyloids of the yeast prions [PSI+], [URE3] and [PIN+] each have an in-register parallel β-sheet architecture. Moreover, we have pointed out that this amyloid architecture can explain how one protein can faithfully transmit any of several conformations to new protein monomers. This explains how proteins can be genes.     

Evolution of budding yeast prion-determinant sequences across diverse fungi

Prions are transmissible self-replicating alternative states of proteins. Four prions ([PSI+], [URE3], [RNQ+] and [NU+]) can be inherited cytoplasmically in Saccharomyces cerevisiae laboratory strains. In the case of [PSI+], there is increasing evidence that prion formation may engender mechanisms to uncover hidden genetic variation. Here, we have analysed the evolution of the prion-determinant (PD) domains across 21 fungi, focusing on compositional biases, repeats and substitution rates. We find evidence for constraint on all four PD domains, but each domain has its own evolutionary dynamics. For [PSI+], the Q/N bias is maintained in fungal clades that diverged one billion years ago, with purifying selection observed within the Saccharomyces species. The degree of Q/N bias is correlated with the degree of local homology to prion-associated repeats, which occur rarely in other proteins (<1% of sequences for the proteomes studied). The evolutionary conservation of Q/N bias in Sup35p is unusual, with only eight other S. cerevisiae proteins showing similar, phylogenetically deep patterns of bias conservation. The [URE3] PD domain is unique to Hemiascomycota; part of the PD domain shows purifying selection, whereas another part engenders bias changes between clades. Also, like for Sup35p, the [RNQ+] and [NU+] PD domains show purifying selection in Saccharomyces species. Additionally, in each proteome, we observe on average several hundred yeast-prion-like domains, with fewest in fission yeast. Our findings on yeast prion evolution provide further support for the functional significance of these molecules.     

Isolation of drugs active against mammalian prions using a yeast-based screening assay

We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs. The method allowed us to identify several compounds effective against budding yeast prions responsible for the [PSI+] and [URE3] phenotypes. These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine. Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay. The compounds isolated here were also active in promoting mammalian prion clearance. These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans.     

Rnq1: an epigenetic modifier of protein function in yeast

Two protein-based genetic elements (prions) have been identified in yeast. It is not clear whether other prions exist, nor is it understood how one might find them. We established criteria for searching protein databases for prion candidates and found several. The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble. The insoluble state is dominant and transmitted between cells through the cytoplasm. When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion [PSI+], the phenotypic and epigenetic behavior of [PSI+] was fully recapitulated. These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.     

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.     

Molecular basis for transmission barrier and interference between closely related prion proteins in yeast

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.