# Genome-wide Translational Changes Induced by the Prion [PSI+]

Agnès Baudin-Baillieu, Rachel Legendre, Claire Kuchly, Isabelle Hatin,

Stéphane Demais, Claire Mestdagh, Daniel Gautheret, Olivier Namy

► To cite this version:

Agnès Baudin-Baillieu, Rachel Legendre, Claire Kuchly, Isabelle Hatin, Stéphane Demais, et al. Genome-wide Translational Changes Induced by the Prion [PSI+]. Cell Reports, 2014, 8 (2), pp.439-448. 10.1016/j CELREP.2014.06.036. hal-04617983

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# Genome-wide Translational Changes Induced by the Prion [PSI<sup>+</sup>]

Agnès Baudin-Baillieu,<sup>1,2,3</sup> Rachel Legendre,<sup>1,2,3</sup> Claire Kuchly,<sup>1,2,4</sup> Isabelle Hatin,<sup>1,2</sup> Stéphane Demais,<sup>1,2</sup> Claire Mestdagh,<sup>1,2,5</sup> Daniel Gautheret,<sup>1,2</sup> and Olivier Namy<sup>1,2,*</sup>

## SUMMARY

Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI<sup>+</sup>] is an aggregated conformation of the translational release factor eRF3. [PSI<sup>+</sup>] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI<sup>+</sup>]–controlled expression remain unknown. We used ribosome profiling in isogenic [PSI<sup>+</sup>] and [psi<sup>-</sup>] strains to identify the changes induced by [PSI<sup>+</sup>]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI<sup>+</sup>]. Surprisingly, [PSI<sup>+</sup>] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI<sup>+</sup>] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi<sup>-</sup>] and [PSI<sup>+</sup>] strains.

## INTRODUCTION

In yeast, prion proteins are composed of regions (called prion-forming domains, PrD) required for the aggregation of the proteins into amyloid fibrils and their subsequent propagation as specific prion variant forms (Bradley and Liebman, 2004). [PSI<sup>+</sup>] is the prion form of eukaryotic release factor 3 (eRF3), which is encoded by the *SUP35* gene in *Saccharomyces cerevisiae* (Ter-Avanesyan et al., 1994). The sequestration of eRF3 in [PSI<sup>+</sup>] aggregates impairs the termination activity of eRF3, thereby increasing stop codon suppression (Liebman and Sherman, 1979) and affecting mRNA stability. It has been suggested that [PSI<sup>+</sup>]-induced plasticity confers a survival advantage in response to environmental fluctuations (True and Lindquist, 2000). Indeed, regions located downstream from the stop codon are not under the same selective pressure as the coding sequence itself and can therefore accumulate mutations more

rapidly. [PSI<sup>+</sup>]-induced readthrough would allow the expression of this part of the mRNA, making it possible to express a new biological function that might be beneficial in a changing environment. Indeed, diverse phenotypes are associated with the presence of [PSI<sup>+</sup>] (Namy et al., 2008; True et al., 2004), but the global landscape of changes in gene regulation in response to weak translation termination efficiency remains to be determined. A recent study aiming to map the loci underlying readthrough-dependent growth phenotypes revealed a complex interplay between the allelic state of the *SKY1* gene (encoding an SR protein kinase (SRPK)) and readthrough-growth phenotypes (Torabi and Kruglyak, 2012). However, the authors of this study used a mutation of the *SUP35* gene to induce stop codon readthrough and to mimic the effect of [PSI<sup>+</sup>], so it is difficult to extrapolate this finding to the physiological consequences of [PSI<sup>+</sup>]. Fitzpatrick et al. (2011) recently investigated SNP variation between two different [PSI<sup>+</sup>] strains. They found that strain 74-D694 harbored a larger number of inactivating stop codon mutation (ISCM) than the other strain (G600). Some of the ISCM detected could potentially account for differences in behavior between nonisogenic [PSI<sup>+</sup>] yeast strains, but they could not explain the physiological consequences of [PSI<sup>+</sup>]. We have previously shown that the *PDE2* stop codon is efficiently read through in the presence of [PSI<sup>+</sup>] (Namy et al., 2002). More recently, we have shown that [PSI<sup>+</sup>] can also stimulate +1 frameshifting (but only when the stimulatory element is a stop codon) at the *OAZ1* gene. This frameshifting event controls the synthesis of the antizyme, which downregulates the polyamine biosynthesis pathway. Half of the known [PSI<sup>+</sup>]-associated phenotypes can be accounted for by the action of [PSI<sup>+</sup>] on the polyamine biosynthesis pathway (Namy et al., 2008). However, our understanding of the overall impact of [PSI<sup>+</sup>] on gene expression remains incomplete, because no genome-wide analysis has yet been published.

We present here a detailed analysis of the yeast [PSI<sup>+</sup>] translome, revealing the important role played by this prion in the regulation of gene expression. As expected, growth medium had a strong impact on the genes affected by [PSI<sup>+</sup>], and we identified more than 100 genes from which alternative proteins were generated by a stop codon readthrough mechanism. All the genes differentially expressed in the [PSI<sup>+</sup>] and [psi<sup>-</sup>]

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<footnote n="1"><sup>1</sup>IGM, University of Paris-Sud, 91405 Orsay, France</footnote>

<footnote n="2"><sup>2</sup>CNRS, UMR 8621, 91405 Orsay, France</footnote>

<footnote n="3"><sup>3</sup>Co-first author</footnote>

<footnote n="4"><sup>4</sup>Present address: Plateforme Génomique, Génopole Toulouse/Midi-pyrénées INRA, 24 Chemin de Borde Rouge, Auzeville CS 52627, 31326 Castanet Tolosan Cedex, France</footnote>

<footnote n="5"><sup>5</sup>Present address: Institute of Molecular Biology AG Roignant Development and RNA Processing, Ackermannweg 4, 55128 Mainz, Germany</footnote>

<footnote>*Correspondence: olivier.namy@igmors.u-psud.fr
http://dx.doi.org/10.1016/j.celrep.2014.06.036</footnote>

<footnote>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).</footnote>
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<figure><img src="image_8.png" /><figcaption>**Figure 1.** Fold Changes in Gene Expression between [PSI⁺] and [psi⁻] Strains</figcaption></figure>

(A) Ribosome densities of two independent replicates were plotted for comparison. Biplot comparing RPKM (reads per million per kilobase of transcript) on a logarithmic scale between two pairs of biological replicate ribosome profiling experiments carried out on two independent [psi⁻] libraries sequenced twice and two [PSI⁺] replicates (R1, R2 respectively).

(B) RPF FC between two [psi⁻] replicates for each of the genes classified in alphabetical order.

(C) Same as in (B) but for the [PSI⁺] strain. The red line indicates the threshold for a 10-fold change.

(legend continued on next page)
isogenic strains were found to be downregulated in [PSI⁺] strains. These genes belong to four principal categories, the most important being stress response genes. Surprisingly, we also found that [PSI⁺] affected reading frame selection, by decreasing translation quality, leading to the production of proteins with a new C terminus. This effect was specific to certain genes involved in translation initiation, tRNA maturation, or amino acid metabolism. Our findings support the notion that the effects of [PSI⁺] are not limited to stop-codon-dependent events and reveal that [PSI⁺] evolved to induce translational errors without stimulating stress response pathways. This fascinating capacity provides a strong adaptive advantage in changing environments. Our results highlight the broader than expected impact of [PSI⁺] on gene expression and its influence on gene expression via various mechanisms.

## RESULTS

Our primary objective was to identify changes in gene expression in the presence of [PSI⁺]. We conducted both a transcriptomic study and ribosome profiling, with isogenic [psi⁻][PIN⁺] and [PSI⁺][PIN⁺] *Saccharomyces cerevisiae* strains in standard growth conditions (see the *Supplemental Experimental Procedures*), to analyze the consequences of [PSI⁺] for gene expression throughout the genome. Moreover, we checked that the effects observed in these conditions were indeed due to [PSI⁺], by performing a ribosome profiling analysis of a [PSI⁺] strain expressing the C-terminal part of Sup35p. The expression of this part of Sup35p in [PSI⁺] counteracts the termination defects due to the aggregation of Sup35p, without curing the cell of [PSI⁺] (Ter-Avanesyan et al., 1993). This constitutes an ideal model for investigations of the consequences of a decrease in translation termination due to the appearance of [PSI⁺]. We adapted the protocol described by Ingolia et al. (2009) to maximize ribosomal RNA depletion. We constructed two independent ribosome protected fragment (RPF) libraries for each strain and sequenced each library twice. Then combined reads to obtain high levels of coding sequence (CDS) coverage for each [psi⁻] and [PSI⁺] strain. In parallel, we generated two independent rRNA-depleted RNA libraries for the [psi⁻] and [PSI⁺] strains. We identified genes influenced by the presence of the prion [PSI⁺], by simultaneously analyzing RPFs and levels of endogenous RNA in [psi⁻] and [PSI⁺] strains.

### [PSI⁺] Represses Large Clusters of Genes

The sequencing of independent libraries of the same strain was highly reproducible, as shown by the Pearson correlation values obtained ($R_1 = 0.9991$ and $R_2 = 0.9992$ for [psi⁻] and [PSI⁺] libraries, respectively; Figure 1A). Translation levels always differed by a factor of less than 10 between two biological replicates (for [psi⁻] or [PSI⁺]; Figures 1B and 1C, respectively), so we

used this value as a threshold for identifying genes with significantly different abundance in RPFs. A comparison of translation levels between [psi⁻] and [PSI⁺] strains identified 75 genes with expression levels differing by a factor of 10–501 between the two strains (Figure 1D; Tables S1A and S1B). The most interesting genes were identified by generating a “volcano” plot showing the fold change (FC) as a function of the adjusted p value (p-adj) for differential expression (Cui and Churchill, 2003). We selected genes with a FC >10 for translation levels (RPF) and with a p-adj <10⁻⁴ (Figure S1; Tables S1A and S1B). A comparison of transcriptomic data from [psi⁻] and [PSI⁺] strains revealed that the main transcriptional consequence of the presence of the prion was a decrease in RNA levels in [PSI⁺] strains (x axis in Figure 1E). Very few genes were more strongly expressed in the presence of the prion, the four largest increases being observed for genes on the 2 µm plasmid (R0020c, R0040c, R0030w, R0010w), suggesting a difference in the copy number of this plasmid between the two strains.

### Most of the Downregulated Genes Belong to the Stress Response Family

We focused our attention on the 75 genes with the largest differences in translation level (>10-fold) between [psi⁻] and [PSI⁺] strains and with a p-adj <10⁻⁴, to ensure that we studied only the most significant changes (red dots on Figure 1E and Tables S1A and S1B). First, we confirmed the genome-wide data by northern blot analyses of eight genes, with an even distribution of RNA FC values. We were unable to detect a signal in [psi⁻] or [PSI⁺] strains for five of these genes (AGX1, GPG1, GSY1, SNO1, XBP1), consistent with the low level of transcription observed for these genes in the transcriptomic data. However, this analysis confirmed the presence of OYE3, YKL071w, and HBN1 mRNA in the polysome fraction of the [psi⁻] strain, whereas these mRNAs were barely detectable in the [PSI⁺] strain (Figure 1F). These mRNAs became detectable in a [PSI⁺] strain cured of the prion by guanidine-HCl treatment or HSP104 overexpression, demonstrating the direct link between [PSI⁺] and the repression of expression for these genes (Figure 1F). For confirmation of the direct involvement of [PSI⁺] in these phenotypes, we compared [PSI⁺] with [PSI⁺] overexpressing SUP35-Cter. We confirmed restoration of termination efficiency, by the observation of a shift from white to red of [PSI⁺] and [PSI⁺]⁺Sup35-Cter colonies, respectively, and by the decrease of stop codon read-through efficiency as previously described (Kabani et al., 2011). Because this overexpression involved the ectopic expression of Sup35p-Cter, we had to shift the cells to a selective medium. Because this shift could potentially modify the pattern of gene expression, we performed ribosome profiling in selective medium, in duplicate, for both [PSI⁺] and [PSI⁺]⁺Sup35p-Cter strains. A high level of reproducibility between replicates was observed, with an R value of 0.98 (Figures S2A and S2B).

(D) RPF FC between [psi⁻] and [PSI⁺] strains. The intervals on the x axis are proportional to the length of the chromosomes.

(E) Biplots show log₁₀-fold differences in RPFs (y axis) and mRNA levels (x axis) between the [psi⁻] and [PSI⁺] strains. Red dots correspond to genes with at least ten times more RPF in the [psi⁻] strain than in the [PSI⁺] strain and a p-adj <1 × 10⁻⁴.

(F) Northern blot analysis of four genes: three are repressed in the [PSI⁺] strain (OYE3, YKL071W, HBN1) and one is used as a control (ACT1), after polysome fractionation on a sucrose cushion. [PSI⁺]HSP104 and [PSI⁺]c corresponds to a [psi⁻] strain obtained by eliminating [PSI⁺] by overexpressing HSP104 or by guanidine HCl treatment, respectively.
<figure><img src="image_9.png" /><figcaption>**Figure 2. Venn Diagram of the Gene Ontology Analysis of Genes Affected by [PSI⁺]**</figcaption></figure>

(A) Functional annotation was carried out in two steps. We first used AMIGO to identify Gene Ontology (GO) terms displaying enrichment in the list. We then manually searched for genes reported to be linked to other biological functions but not present in the GO database (e.g., common regulatory elements). A category is created if at least five different genes are attributed to the same biological pathway or activity. The three categories identified with AMIGO are indicated with a p value score. *A gene involved in prion maintenance: the absence of TSA2 increases the frequency of de novo [PSI⁺] formation.

(B) With the list of genes with translational ambiguities in the [PSI⁺] strain, we used AMIGO software to identify GO term enrichment for biological functions. We used standard settings (i.e., minimum p value = 0.01; minimum number of gene products to define one category: 2). The results were manually curated to reveal near-identical functions. The p value indicated for a group is that provided by AMIGO. We obtained six main categories with a high level of significance; five of these categories are directly linked to the translation process.

Comparisons of the same [PSI⁺] strain grown in rich and selective media showed large differences in gene expression pattern, pre-cluding statistically valid comparisons. However, a comparison of [PSI⁺] and [PSI⁺]⁺Cter in the same growth conditions revealed that 47 genes were downregulated in [PSI⁺], whereas only six genes were upregulated (Figure S2C; Table S1C). This is a very similar to that observed for [PSI⁺] and [psi⁻] in rich medium.

We investigated the functional impact of [PSI⁺], using AMIGO software to search for Gene Ontology term enrichment (Carbon et al., 2009). Three functional categories were particularly abundant among [PSI⁺] downtranslated genes: glycogen biosynthesis, carbohydrate biosynthesis, and oxidation-reduction processes. Using the list of genes (Table S1A), we completed this functional classification by manually sorting all the downregulated genes as a function of either their published physiological functions or their known regulators. This led to the identification of two more biological functions not grouped under GO terms: genes encoding mitochondrial proteins and genes linked to stress response (Figure 2A). These findings indicate that the oxidative and stress response pathways are the main physiological functions impaired by the presence of the [PSI⁺] prion in rich medium. No GO cluster passed the filtering

criteria for the results obtained in selective medium, but several oxidative pathway genes were identified, by eye, in the list.

## [PSI⁺] Generates Translational Ambiguities

Previous ribosome profiling studies have shown that it is possible to map ribosomes to a resolution of one nucleotide, to identify the reading frame translated (Ingolia et al., 2009). This opens up possibilities for detecting the translation of new open reading frames (ORFs). [PSI⁺] is known to affect recoding mechanisms; we were, therefore, interested in determining whether the fidelity of translation was equivalent in [psi⁻] and [PSI⁺] strains. We first looked for known programmed frameshifting (PRF) at the *ABP140*, *EST3*, *OAZ1* genes and stop codon readthrough at the *PDE2* gene. PRF was clearly identified by ribosome profiling for one gene (*ABP140*) (Figure S3), but the low levels of expression of the other genes, together with a low level of recoding (<10%), prevented us from clearly identifying PRF or stop codon readthrough for these genes. We thus searched for genome-wide translational dysfunctions induced by the presence of [PSI⁺]. We retained all genes with an RPKM greater than that for the *ABP140* gene, the only gene for which a known frameshift was detected (Figure S3).
<figure><img src="image_10.png" /><figcaption>**Figure 3. Graphical Representation of Translational Phasing Quality**</figcaption></figure>

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(A) Density of coverage of the A-site position by ribosome-protected fragments (RPF) for the [*psi*⁻] and [*PSI*⁺] strains, shown in blue and green, respectively, for the whole genome. The RPFs are mapped to a composite transcript extending 50 bases on either side of the start (AUG) and stop (UAA) codons. The positions of the initiating and terminating ribosomes are shown at the bottom of the figure. Reads are normalized, with a value of 10 assigned to the highest value. RPFs map mostly to CDS, with a clear increase in their number at the start position. Moreover a clear peak is observed at stop codon position in [*PSI*⁺], highlighting the fact that ribosomes spend more time on stop codons in the [*PSI*⁺] strain.

(B) Graphical representation of the percentage of reads in each of the three possible reading frames. The values for RPF distribution for the [*psi*⁻] and [*PSI*⁺] strains are shown in blue and green, respectively. Reads from the transcriptomic data are shown in purple (only the data for the [*psi*⁻] strain are shown, as the [*psi*⁻] and [*PSI*⁺] strains gave identical results). **p** value <0.01.

(C) All three possible reading frames are represented. Full vertical bars represent stop codons; half bars indicate AUG codons. Frame 1, in which the CDS is usually translated, is shown in blue. The new translated frame is represented in orange. Vertical dotted lines represent the different segments used for the analysis of ribosomal phasing. For each of these segments, the percentage of RPFs in each of the three reading frames is represented by a histogram for both the [*psi*⁻] and [*PSI*⁺] strains. This panel shows data for the *SUI1* gene.

(D) A graphical representation of the construct integrated at the genomic locus is shown in the upper part of the figure. The lower part shows a western blot for the detection of out-of-frame products generated by translational frameshifting at the end of *SUI1* gene in [*PSI*⁺] strain. Lanes 1 and 2 correspond to extracts from the WT (no tag) and the tagged strain, respectively. Equal amounts of protein extract were loaded in each lane. The band at 40 kDa in lane 2 corresponds to the tag translationally fused to Sui1p. No difference is observed in presence of 200 µM MG132 (a proteasome inhibitor, lane 3), demonstrating the stability of the tagged fusion protein.

Most of the RPFs obtained for both [*PSI*⁺] and [*psi*⁻] strains mapped within the CDS with a significant increase in stop codon occupancy within the A-site visible in [*PSI*⁺] (Figure 3A). Moreover, 92% and 77% of RPFs found in frame 1 for [*psi*⁻] and [*PSI*⁺], respectively (this frame corresponding to the initiator AUG), whereas no preferential phase was observed for transcriptomic reads, as expected (Figure 3B). This finding of apparently less accurate phasing in the [*PSI*⁺] strain prompted us to investi-

gate ribosomal phasing in more detail, for each gene, individually. We segmented genes, to obtain segments of equivalent length for analysis. For each segment, we calculated the proportion of RPFs in each of the three reading frames. We retained all genes for which a translation ambiguity (i.e., with at least one segment with less than 60% of RPFs in frame 1) was detected. We identified 14 and 119 genes with at least one translational ambiguity in the [*psi*⁻] and [*PSI*⁺] strains, respectively (Tables S1 and S2).
These data indicate a broader than expected impact of [PSI⁺] on gene expression. However, translation ambiguities were limited to a subset of genes, suggesting a specific mechanism.

## Translation Ambiguities Are Not Restricted to Stop-Codon-Dependent Events

Surprisingly, these translational ambiguities occur evenly along the length of the CDS (Table S3), suggesting that these events are not a direct consequence of the low efficiency of stop codon recognition for these genes in a [PSI⁺] background. Genes with translational ambiguities in [PSI⁺] strains were found to belong mostly to three major functional categories: glucose catabolic process, translation initiation, and carboxylic acid metabolic process. (Figure 2B). The SUI1 gene, encoding translation initiation factor eIF1, displayed one of the most significant translational ambiguities, with 70% of the ribosomes in the alternative reading frame and only 28% of ribosomes in the AUG reading frame (Table S3; Figure 3C). One key question concerns the origin of these out-of-frame footprints: do they come from ribosomal frameshift or from the initiation (or reinitiation) of translation within the CDS by a new ribosome? We addressed this question by inserting a 13myc tag immediately downstream from the SUI1 stop codon, in the –1 reading frame, at the chromosomal locus in the [PSI⁺] strain. Western blots clearly showed a single band at 40 kDa, a size consistent with a translational fusion between eIF1 and the –1 reading frame overlapping the end of the SUI1 gene. This protein was present in similar amounts in the presence and absence of MG132, a proteasome inhibitor (Figure 3D). This suggests that a translational frameshift occurs close to the end of the SUI1 gene, resulting in the production of a stable protein with an alternative C terminus. We found no difference in translation ambiguities between [PSI⁺] and [PSI⁺]⁺Cter, indicating that these ambiguities were not directly linked to the translation termination deficiency in [PSI⁺]. Thus, [PSI⁺] can affect translation fidelity in various ways, independently of its effect on stop codon recognition.

## [PSI⁺] Causes Genome-wide Translational Stop Codon Readthrough

Given the physiological role of eRF3, we would expect most of the translational defects associated with [PSI⁺] to be linked to stop codons. A short list of genes with ribosome footprints downstream from the stop codon of the CDS has recently been reported (Dunn et al., 2013). Our data indicated that 68 and 27 genes displayed such footprints downstream from the stop codon in the [PSI⁺] and [psi⁻] strains, respectively (Table S4). We found no overlap with the previously published list of readthrough candidates. This may reflect differences in genetic background. Such footprints might correspond to nontranslating ribosomes that are not recycled, but the much larger number of genes displaying readthrough in [PSI⁺] suggests that they correspond to bona fide stop codon readthrough events. For confirmation, we analyzed the readthrough candidates identified by ribosome profiling in [PSI⁺] cells overexpressing the carboxy-terminal part of Sup35p. We reasoned that all stop codon read-through events due to a defective termination process in the [PSI⁺] strain would be abolished in this condition. We found 205 and 166 genes with stop codon readthrough patterns in

[PSI⁺] and [PSI⁺]⁺Cter, respectively. Only the 140 candidate genes with a potential extension longer than a RPF (i.e., 30 nt) were conserved for further investigations (Table S5). This list included 111 genes (80%) displaying a partial or complete reduction of ribosomal footprints downstream from the stop codon in Sup35p-Cter overexpression, which thus corresponded to bona fide readthrough events (Table S5). For the other 29 genes, we concluded that the ribosome footprints downstream from the stop codon were probably not due to a stop codon readthrough event.

## DISCUSSION

### Gene Repression Promoted by [PSI⁺]

Ribosome profiling data can be analyzed both quantitatively and qualitatively. Careful quantitative analysis of our data clearly indicated that 75 genes were strongly repressed (more than 10-fold) in presence of [PSI⁺] in rich medium. This repression was also observed in our transcriptomic analysis (Figure 1E) and was confirmed by northern blotting (Figure 1F). Prion elimination by treatment with guanidine-HCl or HSP104 overexpression led to the synthesis of these mRNAs, demonstrating the epigenetic repression of these genes by [PSI⁺].

It has been hypothesized that [PSI⁺] could modify gene expression through translation-independent mechanisms. Indeed, mass spectrometry analyses of [PSI⁺] aggregates have identified a large number of other proteins associated with the aggregate (Nevzglyadova et al., 2009). [PSI⁺] aggregates may prevent these proteins from playing their physiological roles, through sequestration. We addressed this question and determined whether at least some of the observed phenotypes were due to a decrease in translation termination efficiency, by constitutively expressing the carboxy-terminal part of Sup35 in a [PSI⁺] strain.

This strain constitutes an ideal model for determining which [PSI⁺]-associated phenotypes are due to a translation termination defect. However, the shift to a selective medium had a very strong impact in gene expression, precluding comparison of the results obtained with those obtained in the previous set of conditions. Despite this limitation, we found that 47 genes were less expressed in [PSI⁺] than in [PSI⁺]⁺Cter (same translation termination efficiency as [psi⁻]), in comparable growth conditions (Figure S2; Table S1C). These results are reminiscent of those obtained for [PSI⁺] and [psi⁻] strains in rich medium, in which 100 genes were found to be less expressed in [PSI⁺] than in [psi⁻]. Although we cannot not totally exclude the possibility that Sup35-Cter does not fully rescue genome-wide termination efficiency, we can draw two important conclusions from these results. (1) Growth conditions have a strong impact on the effect of [PSI⁺] on gene expression, confirming published genetic data for [PSI⁺] (Namy et al., 2008; True and Lindquist, 2000). (2) Most of the underexpression observed is directly linked to [PSI⁺], because it was affected by the expression of Sup35p-Cter. It is not currently possible to determine whether a decrease in ribosome load leads to mRNA destabilization or whether the apparent decrease in translation simply reflects transcriptional repression.

We found that 30 (40%) of the genes repressed in the presence of [PSI⁺] were linked to oxidative pathways, and 37
[PSI⁺]

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[PSI⁺] + Cter

<figure><img src="image_13.png" /><figcaption>**Figure 4. Visualization of Stop Codon Read-through Events**</figcaption></figure>

The CDS of TOM5 is indicated by a gray box. Below, ribosomal footprint density along the length of the CDS is represented with chromosomal coordinates. A higher magnification of the extension region (i.e., downstream from the stop codon of the CDS) is shown, for the visualization of ribosome density in this area. A sharp decrease in ribosome density is visible after the stop codon, but ribosomes are still clearly detectable. There is also a difference in the density of ribosomes loaded on the extension segment between the [PSI⁺] (left) and the [PSI⁺]⁺Cter (right) strain, with RPKM values in the extension of 0.19 and 0.06, respectively.

(50%) were linked to stress responses in yeast (Figure 2A). In total, 18 genes were common to these two groups (Figure 2A). Thus, the stress response and oxidative pathways are the principal physiological functions affected by the presence of the [PSI⁺] prion. The identification of such a large number of genes involved in stress and metabolic pathways is consistent with the phenotypes observed to be associated with [PSI⁺]. Indeed, [PSI⁺] is known to modify yeast metabolism and to have positive or negative effects on yeast growth under conditions of stress (Namy et al., 2008). Moreover oxidative pathways are well known to be strongly linked to [PSI⁺] physiology (Sideri et al., 2010, 2011). We carried out ribosome profiling in standard growth conditions, but our findings nonetheless provide a general view of the overall changes in gene expression, potentially accounting for the appearance of so many phenotypes in stress conditions. We cannot yet identify the initial event leading to this downregulation of the expression of about 100 genes in [PSI⁺] cells, but we can already draw the fascinating conclusion that [PSI⁺] shuts off the expression of many genes involved in the stress response.

This finding was unexpected, because [PSI⁺] formation is induced by oxidative stress (Chernoff, 2007) and [PSI⁺] has been shown to confer a growth advantage under stress conditions. We therefore expected [PSI⁺] to induce the expression of genes involved in the stress response. However, it should be borne in mind that most [PSI⁺]-associated phenotypes are detrimental, and that this was long seen as evidence for considering [PSI⁺] to be a “laboratory disease.” Our results may provide a molecular explanation for some of the detrimental effects of [PSI⁺].

### [PSI⁺] Causes Genome-wide Stop Codon Readthrough

The first molecular consequence of [PSI⁺] is a decrease in translation termination efficiency. However, the analysis of stop codon readthrough is complicated by the high efficiency of the translation termination reaction, even in the presence of [PSI⁺]. This results in a small number of footprints downstream from the stop codon. We defined genes with unambiguous ribosome footprints downstream from the official stop codon, as previously described (Dunn et al., 2013). We identified 68 and 205 stop codon readthrough events in [PSI⁺], in rich and selective

media, respectively (Table S4). Interestingly, the genes displaying a readthrough pattern differed between growth media, indicating that stop codon readthrough is not a constitutive event, instead being strongly affected by environmental factors. This clearly adds a layer of regulation to gene expression according to the growth conditions. Interestingly, 80% of these stop codon readthrough events were less frequent in [psi⁻] or [PSI⁺]⁺Cter, demonstrating a direct link between these events and the translation termination defect in [PSI⁺], whatever the growth conditions (Figure 4; Tables S4 and S5).

We found ribosome footprints downstream from the stop codons of 20% of the genes even if the C-terminal part of Sup35 was overexpressed. There are two possible reasons for this. (1) These stop codon readthrough events are not sensitive to Sup35-Cter overexpression. This possibility is supported by a previous report showing that Sup35 mutations cause readthrough at some stop signals but not at others (Salas-Marco and Bedwell, 2004). (2) These footprints are generated independently of termination efficiency, through a failure of posttermination ribosome recycling, for example, as recently described (Guydosh and Green, 2014).

### [PSI⁺] Generates Translational Ambiguities

One of the most remarkable findings of this study is that 119 genes show translational ambiguities in the [PSI⁺] strain (Table S3), eight times as many as in the [psi⁻] strain (Table S2), clearly indicating a major role for [PSI⁺] in translation accuracy. However, the lack of difference in translational ambiguities between [PSI⁺] and [PSI⁺]⁺Cter indicates that these ambiguities are not due to the low translation termination efficiency in [PSI⁺]. This clearly suggests that the effects of [PSI⁺] extend beyond a simple defect in translation termination. The ability of the N-terminal part of eRF3 to induce a prion conformation has been retained over a period of at least 500 million years, so this ability to promote translational ambiguities must clearly be advantageous in [PSI⁺] strains. Furthermore, the ability of [PSI⁺] to induce the expression of new regions of the yeast genome is often presented as a strong advantage, allowing the yeast to express new phenotypes that may enable it to adapt to fluctuating environments. The generation of a low, but significant level of translational ambiguity may fulfill this function.
## Could Another Prion Be Responsible for These Phenotypes?

One of the major difficulties involved in the interpretation of data concerning prion phenotypes and physiological roles concerns the interplay between prions. Indeed, the presence of one prion increases the probability of another prion appearing (Derkatch et al., 2001). This has been shown for [PSI⁺]⁴, which is required for the appearance of [PSI⁺] (Tuite et al., 2008). We checked that the changes observed in [PSI⁺] cells were not due to another prion, by using Sup35p-Cter overexpression to restore translation termination efficiency to wild-type levels. This made it possible to show that both the observed downregulation and stop codon readthrough were direct consequences of the low translation termination efficiency in [PSI⁺]. However, translational ambiguities were not affected by the expression of Sup35p-Cter, suggesting that they are due either to an unknown property of [PSI⁺] or to another prion. Clearly, the hypothesis that the translational ambiguities observed in this study are directly due to an unknown effect of the [PSI⁺]⁴ prion merits further analysis.

## How Can [PSI⁺] Have Such a Complex Effect on Gene Expression?

One major question is that of whether the observed differences between [psi⁻] and [PSI⁺] are due to a direct effect of [PSI⁺] on each of the genes concerned, or whether there is a general regulator, controlling all these genes, that is affected by [PSI⁺]. The presence of [PSI⁺] has three main consequences: (1) a decrease in gene expression, (2) the induction of translational ambiguities, and (3) the stimulation of stop codon readthrough. [PSI⁺] has generally been shown to have a significant, but moderate effect on stop codon recognition, and we confirmed this view by identifying a large set of genes displaying translational readthrough in [PSI⁺] (Table S3). However, these genes are not underexpressed, so we conclude that most of these translational decreases are an indirect consequence of the action of [PSI⁺] on some genes. So what are the primary targets of [PSI⁺]? It is difficult to answer this question on the basis of the observed downregulation, unless we can identify a mutation mimicking the gene repression induced by [PSI⁺].

Another interesting question concerns the way in which [PSI⁺] generates translational ambiguities, equally distributed throughout the CDS. The presence of translational ambiguities in regions at some distance from the stop codon was unexpected, because many mechanisms of action have been hypothesized for [PSI⁺] on the basis of the function of eRF3, but a direct action of [PSI⁺] on reading frame selection was considered unlikely. These translational ambiguities were resistant to the overexpression of Sup35⁺Cter, indicating that they were not a consequence of the decrease in translation termination efficiency. One may suggest that some of these translational ambiguities are due to the translation of alternative mRNA forms not detectable in [psi⁻]. Another possible explanation was provided by an analysis of genes displaying translation ambiguities. One of the most important ambiguities was that found for the SUI1 gene, for which 70% of the ribosome footprints were not in the initial reading frame (Figure 3C). We were able to confirm these findings by tagging, at the chromosomal locus, the peptide

expressed by this alternative reading frame. The western blot (Figure 3D) showed a single band in the tagged [PSI⁺]⁴ strain, at 40 kDa, corresponding to a fusion of the entire eIF1 protein and the tag in the –1 frame. Whatever the mechanism involved, this finding suggests a link between eIF1 gene and [PSI⁺]. This finding is of particular interest, because eIF1 is involved in the selection of translation initiation sites (Donahue et al., 1988). This protein is known to play a crucial role in translation start site selection and translation elongation; modifications of this protein could greatly decrease translation accuracy (Yoon and Donahue, 1992). This would result in defective AUG recognition, allowing ribosomes to scan downstream from the initial AUG codon to initiate translation in an alternative frame. This probably reflects the situation for the *CRP1* gene (Figure S4), for which more than 40% of the ribosome footprints were located in frame with the second AUG found in the mRNA. In our model, in presence of [PSI⁺], SUI1 would affect translation accuracy for a large number of genes. However, it remains unclear how these translational defects are restricted to genes linked to translation initiation and to the metabolism of tRNAs and amino acids.

It has been shown that low levels of mistranslation activate the unfolded protein response and environmental stress response pathways, leading to a preadaptation to stress that may provide cells with a selective advantage under certain environmental conditions (Santos et al., 2004). [PSI⁺], which also promotes translational errors in a large number of genes, might also be expected to induce a stress response. However, our results indicate that the opposite is true, with the prion actually repressing genes involved in the stress response. So how can we reconcile these findings with the initial hypotheses? If a decrease in translation fidelity was responsible for triggering stress responses, this might impair the fitness of [PSI⁺]⁴ strains in the long term, by limiting the potential advantage conferred by the production of new proteins, preventing [PSI⁺]⁴ strains from adapting to a new environment. On the basis of these assumptions, we propose a model describing the two modes of action of [PSI⁺], which stimulates translational errors and simultaneously prevents induction of the stress response. This might constitute a very interesting case of coevolution allowing [PSI⁺] to be advantageous. This is an important issue that will undoubtedly be addressed by future studies.

In conclusion, we show that two categories of events are generated by [PSI⁺]: (1) translational ambiguities, independent of the translation termination defect due to [PSI⁺], suggesting that the amyloid aggregates sequester other proteins involved in the control of translation quality; and (2) stop codon readthrough and the downregulation of about 100 genes, as a direct consequence of the low termination efficiency caused by [PSI⁺]. Further translatome-wide analysis in stress conditions or in different genetic backgrounds, in the presence and absence of [PSI⁺], should increase our understanding of the multistep effects of this prion on gene expression.

## EXPERIMENTAL PROCEDURES

### *Yeast Manipulation and Standard Molecular Biology*

Yeast strain 74-D694 (mata ade1-14 [UGA] ura3-52 trp1-289 [UAG] his3Δ200 leu2-3,112 [psi⁻][PIN⁺]) with [PSI⁺] or without [psi⁻] the prion form of the
Sup35p termination factor was grown to an OD₆₀₀ of 0.6-0.8 at 30°C in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose).

Total RNA for northern blots was extracted by the same procedure used for the preparation of RPF libraries, except for the use of RNase I. After polysome fractionation on a sucrose cushion, RNA was hybridized with a ³²P probe corresponding to the targeted gene.

### Ribosome Profiling and Data Analysis

For the generation of ribosome and mRNA profiling libraries, we used [psi⁻] and [PSI⁺] yeast strains (74-D694) both of which were [PIN⁺]. RPF libraries were prepared according to the established protocol (Ingolia et al., 2009), with the use of primers to eliminate major rRNA contamination (see the Supplemental Experimental Procedures), and sequenced by high-throughput methods (Illumina). We defined coordinates for each active gene in our yeast strains, taking the UTR into account. We used unique reads from the combined libraries to define “transcriptional units” (TU). The sum of reads for each TU was then quantified and normalized with DESeq (Anders and Huber, 2010) for each [psi⁻] and [PSI⁺] RPF library. These values were then used to compare levels of gene expression between the two strains. Differential gene expression was determined with HTSeq-count and read counts were normalized with DESeq R library. Qualitative analyses of ribosome positions were carried out by calculating RPF density at the P-site codon with 28-mer RPFs.

### mRNA Fragment Preparation for Transcriptome Analysis

Total RNA was extracted from 100 ml YPD culture (OD = 0.8) by the hot acid phenol method. The Ribominus Eukaryote kit (Invitrogen) was used to eliminate rRNA from 10 mg of total RNA, according to the manufacturer's instructions. The resulting RNA preparation was fragmented by Zn²⁺ treatment.

### Library Preparation

Directional RNA-seq library preparation was carried out with the Small RNA Sample Prep Kit and the v1.5 sRNA 3' Adaptor (Illumina), according to the manufacturer's protocol, and the library was checked with the bioanalyzer small RNA kit.

### Reproducibility of Experiments

We estimated reproducibility (Figures 1 and S2), by measuring expression as RPKM (reads per kilobase per million reads), because this accounts only for relative, intrasample differences in gene expression, hence providing a more conservative estimate of reproducibility than DeSeq normalization. The linear regression plot (Figure 1) between RP and TP was generated with the R package.

### Functional Classification of Genes

We investigated the molecular mechanisms underlying gene repression and translational ambiguities, by sorting the identified genes with the Gene Ontology database (v1.8) and AMIGO (Boyle et al., 2004).

### Detection of Alternative Proteins by Western Blotting

A BgIII-HindIII fragment (1,230 bp) containing the *SUI1* gene was inserted between the BamHI and HindIII sites of the pFL39 plasmid (Bonneaud et al., 1991). This plasmid was introduced into the [*PSI⁺*] strain such that the insertion of the tag at the chromosomal locus would not affect the expression of *SUI1* (a complete deletion of *SUI1* is lethal). The strain was transformed with a PCR product tagging of the -1 reading frame overlapping the end of the *SUI1* gene with a 13Myc epitope (Longtine et al., 1998). Western blots were carried out on total extracts of cells grown at 30°C in minimum medium. An aliquot of the culture was treated for 2 hr with the proteasome inhibitor MG132, at a concentration of 200 mM (Sigma-Aldrich). The 13Myc tag was detected with the 9E10 rat anti-Myc primary antibody (Roche) and the rabbit anti-rat IgG HRP-conjugated secondary antibody (Sigma). Proteins were visualized with Amersham ECL Prime Western Blotting Reagents (GE Healthcare).

### ACCESSION NUMBERS

The complete set of data has been deposited to the NCBI Gene Expression Omnibus under accession number GSE41590.

## SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, four figures, and five tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.06.036.

## AUTHOR CONTRIBUTIONS

A.B.-B., I.H., C.M., and O.N. designed experiments and set up the ribosome profiling. R.L., C.K., and D.G. performed the bioinformatics and statistical analysis. S.D. performed northern blots. All the authors discussed the results and participated in the writing of the manuscript.

## ACKNOWLEDGMENTS

English usage was corrected by Alex Edelman & Associates. This work benefited from the facilities and expertise of the IMAGIF high-throughput sequencing platform (Centre de Recherche de Gif, www.imagif.cnrs.fr). The bioinformatics analysis was performed by the e-bio platform (http://ebio.u-psud.fr/). We would like to thank J.P. Rousset for critical reading of the manuscript. This work and R.L. are supported by a grant from the ANR (ANR-2011-BSV6-011) to O.N.

Received: October 16, 2013

Revised: April 14, 2014

Accepted: June 19, 2014

Published: July 17, 2014

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