The Spontaneous Appearance Rate of the Yeast Prion [PSI+] and Its Implications for the Evolution of the Evolvability Properties of the [PSI+] System

Epigenetically inherited aggregates of the yeast prion [PSI+] cause genomewide readthrough translation that sometimes increases evolvability in certain harsh environments. The effects of natural selection on modifiers of [PSI+] appearance have been the subject of much debate. It seems likely that [PSI+] would be at least mildly deleterious in most environments, but this may be counteracted by its evolvability properties on rare occasions. Indirect selection on modifiers of [PSI+] is predicted to depend primarily on the spontaneous [PSI+] appearance rate, but this critical parameter has not previously been adequately measured. Here we measure this epimutation rate accurately and precisely as 5.8 × 10⁻⁷ per generation, using a fluctuation test. We also determine that genetic “mimics” of [PSI+] account for up to 80% of all phenotypes involving general nonsense suppression. Using previously developed mathematical models, we can now infer that even in the absence of opportunities for adaptation, modifiers of [PSI+] are only weakly deleterious relative to genetic drift. If we assume that the spontaneous [PSI+] appearance rate is at its evolutionary optimum, then opportunities for adaptation are inferred to be rare, such that the [PSI+] system is favored only very weakly overall. But when we account for the observed increase in the [PSI+] appearance rate in response to stress, we infer much higher overall selection in favor of [PSI+] modifiers, suggesting that [PSI+]-forming ability may be a consequence of selection for evolvability.THE yeast phenotype [PSI+] is characterized by prion aggregates of the protein Sup35. Cells are in either a [psi−] (normal) or [PSI+] state, depending on the absence or presence of the prion aggregates (Figure 1, a and b). Sup35 prion aggregates replicate in a similar fashion to mammalian prions but are cytoplasmic and, as such, the prion state is cytoplasmically inherited (Wickner et al. 1995).Open in a separate windowFigure 1.—Comparison between the three possible modes ([PSI+], genetic mimic, point mutation revertant) of the expression of 3′-UTR sequences in yeast. (a) The normal [psi−] phenotypic state; (b) the [PSI+] prion causes readthrough and low-level expression of 3′-UTRs across multiple genes, appearing at rate m_PSI; (c) a genetic mimic of [PSI+] such as the sal3-4 mutant of Sup35 (Eaglestone et al. 1999) appearing at rate m_mimic not reversible by the application of guanidine hydrochloride; (d) a point mutation in a single stop codon at rate μ_point, leading to incorporation of formerly 3′-UTR into a single coding sequence. (e) [PSI+] can act as a “stop-gap” mechanism, buying a lineage more time to acquire one or more adaptive stop codon readthrough point mutations. When this genetic assimilation is complete, [PSI+] can revert to [psi−] (Masel and Bergman 2003; Griswold and Masel 2009).When not part of an aggregate, Sup35 helps mediate translation termination in yeast (Stansfield et al. 1995b; Zhouravleva et al. 1995). Sup35 molecules that are incorporated into nonfunctional prion aggregates are presumably not available for translation termination, which can lead to the translation of stop codons by near-cognate tRNAs (Figure 1b) (Tuite and Mclaughlin 1982; Pure et al. 1985; Lin et al. 1986). This partial loss of Sup35 function leads to an increased frequency of readthrough translation of 3′-untranslated regions (3′-UTR) across all genes (Figure 1b). This increase is modest in wild-type yeast, from an average readthrough rate of 0.3% in [psi−] cells up to 1% in [PSI+] cells (Firoozan et al. 1991). Some [PSI+] yeast strains grow faster than [psi−] controls in certain harsh environments, suggesting that readthrough translation of some 3′-UTRs may be adaptive in certain conditions (True and Lindquist 2000; Joseph and Kirkpatrick 2008). This directly shows that [PSI+]-mediated capacitance may increase evolvability in the laboratory. [PSI+]-mediated phenotypes have a complex genetic basis, involving multiple loci (True et al. 2004).As an epigenetically inherited protein aggregate, [PSI+] can easily be lost after some generations (Cox et al. 1980). This returns the lineage to its normal [psi−] state and restores translation fidelity. If a subset of revealed phenotypic variation is adaptive, it may have lost its dependence on [PSI+] by this time (True et al. 2004). This process of genetic assimilation may, for example, involve one or more point mutations in stop codons, increasing readthrough up to 100% (Figure 1e) (Griswold and Masel 2009). This leaves the yeast with a new adaptive trait and with no permanent load of other, deleterious variation.In general, stop codons can be lost either directly through point mutations or indirectly through upstream indels. This leads to novel coding sequence coming from in-frame and out-of-frame 3′-UTRs, respectively. [PSI+] is expected to facilitate only the former, while mutation bias favors the latter. Yeasts show a much higher ratio of in-frame to out-of-frame 3′-UTR incorporation events than mammals do (Giacomelli et al. 2007), confirming a role for [PSI+] in capacitance-mediated evolvability in natural populations.The adaptive evolution both of evolvability in general (Sniegowski and Murphy 2006; Lynch 2007; Pigliucci 2008) and of capacitance in particular (Dickinson and Seger 1999; Wagner et al. 1999; Partridge and Barton 2000; Brookfield 2001; Pal 2001; Meiklejohn and Hartl 2002; Ruden et al. 2003) is highly controversial. In general, any costs of evolvability are borne in the present, while the benefits lie in the future, making it difficult for natural selection to favor an evolvability allele. For example, mutation rates seem to be set according to a trade-off between metabolic cost (favoring higher mutation rates) and the avoidance of deleterious effects (favoring lower mutation rates) (Sniegowski et al. 2000). The fact that mutation creates variation, the ultimate source of evolvability, is merely a fortuitous consequence of the metabolic cost of fidelity.Previous theoretical population genetic studies have, however, suggested that modifier alleles promoting the formation of [PSI+] might, unlike mutator alleles, be favored for their evolvability properties (King and Masel 2007; Masel et al. 2007; Griswold and Masel 2009; Masel and Griswold 2009). These models depend, however, on a number of parameter estimates. In particular, a number of predictions depend on the spontaneous rate of [PSI+] formation (Masel and Griswold 2009).

[PSI+] appearance rates and the fluctuation test:

The most widely cited spontaneous appearance rate for [PSI+] is m_PSI ∼ 10⁻⁷–10⁻⁵, on the basis of experiments by Lund and Cox (1981). This estimate was calculated as the proportion of colonies scored as [PSI+] after growth over multiple generations from a single founding [psi−] clone. If [PSI+] happens to appear in the first generation of growth, this leads to a “jackpot” event with only one switching event, but many [PSI+] colonies. The proportion of colonies scored as [PSI+] therefore yields a systematic overestimation of the [PSI+] appearance rate.Various implementations of the fluctuation test (Luria and Delbrück 1943) can address such effects. The mutation rate experiment is replicated many times using independent populations, and a Luria–Delbrück distribution is fitted to the results across all replicates. In a simulation study, Stewart (1994) examined a number of estimators of the underlying Luria–Delbrück distribution and found that the maximum-likelihood estimator performed the best.Originally developed to study mutation rates, the fluctuation test can also be used for estimating epimutation rates. Fluctuation tests have been used to estimate the rate of gene silencing in Chinese hamster ovary cells (Holliday and Ho 1998) and in the yeast Schizosaccharomyces pombe (Singh and Klar 2002). However, fluctuation tests do not appear to be used routinely for epimutation rate estimates. For example, although the rates of spontaneous appearance and disappearance of [ISP+], a prion-like element in yeast, have been measured using the fluctuation test (Volkov et al. 2002), to the best of our knowledge there are no published estimates of the spontaneous rate of [PSI+] appearance as measured using a fluctuation test. Although results from the fluctuation test can be confounded by reverse epimutation, or back-switching, this is an issue only if the rate of back-switching is very high, e.g., 10⁻¹–10⁻² per generation (Saunders et al. 2003). This is not the case for [PSI+], for which the reverse epimutation rate (loss of [PSI+]) is <2 × 10⁻⁴ (Tank et al. 2007).

Other [PSI+]-like phenotypes, including genetic mimics:

[PSI+] causes partial loss of Sup35 function, leading to elevated rates of translational readthrough at all stop codons (Figure 1b). There are many other spontaneous changes, presumably mutations, that also lead to elevated translational readthrough (Lund and Cox 1981). Mutations that affect readthrough at all stop codons (Figure 1c) (sometimes called “[PSI+]-like”) can be considered as genetic “mimics” because they produce the same phenotype as the Sup35 aggregate, but are generally not epigenetically inherited. A specific example of such a genetic mimic was characterized by Eaglestone et al. (1999), who identified the sal3-4 point mutation in the SUP35 gene. This leads to a defect in the Sup35 protein structure rendering the termination process less efficient (Eaglestone et al. 1999). The sal3-4 mutant can therefore be considered a partial loss-of-function genetic mimic of [PSI+], since it generates the same readthrough phenotype. Translation termination could also potentially be impaired through other point mutations or deletions, for example, in either the SUP35 or the SUP45 gene (Stansfield et al. 1995a) or in a tRNA that mutates to recognize stop codons at a higher rate. The presence of genetic mimics, whose effects are less reversible than those of [PSI+], can affect the evolution of the evolvability properties of the [PSI+] system such as its epimutation rate (Lancaster and Masel 2009). Note that genetic mimics are quite different from much rarer point mutations that convert stop codons into coding sequence (Figure 1d), resulting in readthrough at a single gene rather than multiple genes.Here we performed experiments to obtain accurate and precise estimates of the baseline appearance rates of both [PSI+] and [PSI+]-like phenotypes in permissive laboratory conditions, excluding stop codon point mutations that affect only a single gene. Our estimates are superior to previous estimates, since we use the fluctuation test. We consider the consequences of these estimates for the evolution of the [PSI+] system.