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Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity Graphical Abstract

Authors Danny D. Nedialkova, Sebastian A. Leidel

Correspondence [email protected]

In Brief Optimal codon translation rates— ensured by the presence of nucleoside modifications in the tRNA anticodon—are critical for maintaining proteome integrity.

Highlights d

tRNA anticodon modification loss slows translation at cognate codons in vivo

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Codon-specific translational pausing triggers protein misfolding in yeast and worms

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Codon translation rates and protein homeostasis are restored by tRNA overexpression

Nedialkova & Leidel, 2015, Cell 161, 1606–1618 June 18, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.cell.2015.05.022

Accession Numbers GSE67387

Article Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity Danny D. Nedialkova1,2 and Sebastian A. Leidel1,2,3,* 1Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany 2Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany 3Faculty of Medicine, University of Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2015.05.022 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress, accumulate aggregates of endogenous proteins, and are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA modifications in maintaining proteome integrity.

INTRODUCTION The rate of peptide synthesis by ribosomes is not uniform along mRNAs and is often punctuated by transient pauses. This can impact protein folding, as proteins begin to attain their active conformations in concert with their synthesis (Pechmann et al., 2013). In particular, the differential use of synonymous codons may define local elongation speed and, in turn, aid cotranslational protein folding (Komar, 2009; Pechmann and Frydman, 2013; Thanaraj and Argos, 1996). Though synonymous substitutions in individual genes can indeed have detrimental effects on protein function (Kimchi-Sarfaty et al., 2007; Xu et al., 2013; Zhou et al., 2013), the factors that control translation rates at individual codons in vivo are poorly understood 1606 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

(Tarrant and von der Haar, 2014), and evidence linking global changes in these rates to protein misfolding in living cells is so far lacking. A key factor modulating codon translation rates is tRNA selection. During each round of peptide elongation, the ribosome decodes the mRNA codon in its acceptor (A) site by selecting a matching (cognate) aminoacyl-tRNA from a large pool of tRNA molecules. The abundance of the cognate aminoacyl-tRNA and competing near-cognate ones, the cellular demand for a tRNA species, and the nature of codon-anticodon base pairing all impact the speed of decoding (Fluitt et al., 2007; Pechmann and Frydman, 2013; Stadler and Fire, 2011; Varenne et al., 1984). Within the ribosomal A site, cognate codon-anticodon interactions are identified based on Watson-Crick base pairing. The third codon position can form non-standard (wobble) pairs, allowing some tRNAs to be used in decoding multiple codons. In all kingdoms of life, the tRNA wobble nucleoside (position 34) frequently carries chemical modifications that enhance codon binding in vitro (Agris et al., 2007). The most striking example of this is the wobble uridine (U34), which is almost always modified (Grosjean et al., 2010), and the genes required for this in eukaryotes are conserved from yeast to mammals (Glatt and Mu¨ller, 2013; Shigi, 2014). Loss of U34 modifications decreases fitness through poorly defined mechanisms. Although deletion of genes dedicated to modifying U34 does not reduce tRNA stability or aminoacylation (Johansson et al., 2008), it is embryonic lethal in flies and mice (Chen et al., 2009b; Walker et al., 2011), decreases resistance to challenging environments in yeast (Dewez et al., 2008; Huang et al., 2005; Leidel et al., 2009), and causes developmental and neuronal dysfunction in nematodes (Chen et al., 2009a). In humans, genetic variants and loss-of-function mutations within the orthologous genes are associated with neurodegeneration and intellectual disabilities (reviewed in Torres et al., 2014). A number of observations suggest that cellular dysfunction upon loss of U34 modifications may be tied to inefficient translation via a subset of tRNAs with modified U34. In eukaryotes, the U34 base of 11 cytoplasmic tRNAs carries a 5-methoxycarbonylmethyl (mcm5) or 5-carbamoylmethyl (ncm5). The addition of these moieties requires the six-subunit Elongator (Elp) complex (Huang et al., 2005; Johansson et al., 2008) (Figure 1A). Following mcm5U34 addition in three species—tEUUC, tKUUU,

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Figure 1. Loss of U34 Modifications Leads to Codon-Specific Ribosome Pausing in Yeast and Nematodes (A) Pathways for wobble uridine (U34) modification in the eukaryotic cytoplasm.

and tQUUG—the base is further decorated with a 2-thio group (s2) via a sulfur-relay pathway that requires Uba4, Urm1, Ncs2, and Ncs6 (Leidel et al., 2009; Nakai et al., 2008; Schlieker et al., 2008) (Figure 1A). Thiolation of U34 in tEUUC, tKUUU, and tQUUG is universally conserved, forming a part of the core translational apparatus in extant cells (Grosjean et al., 2014). Several studies have hinted at a disproportionate functional importance of U34 modifications in these three isoacceptors. The stress-induced growth defects of yeast lacking Elp subunits are nearly identical to those observed upon loss of U34 2-thiolation (Nakai et al., 2008) and can be rescued by increasing cellular levels only of species that normally carry mcm5s2U34: tEUUC, tKUUU, and tQUUG (Bauer et al., 2012; Bjo¨rk et al., 2007; Dewez et al., 2008; Esberg et al., 2006; Leidel et al., 2009). Long repeats of the codons pairing with these three tRNAs (GAA, AAA, and CAA) decrease protein output from highly expressed reporter genes in urm1D yeast (Rezgui et al., 2013), and ribosomes accumulate at these codons in ncs6D and uba4D yeast grown under nutrient-replete conditions (Zinshteyn and Gilbert, 2013). The physiological relevance of this codon-specific ribosome pausing is unclear, given that the mutants show little growth impairment under such conditions (Huang et al., 2005; Leidel et al., 2009). In contrast, their growth is severely impaired by environmental stress, such as exposure to the sulfhydryl-oxidizing agent diamide, or TOR pathway inhibition by rapamycin (Bjo¨rk et al., 2007; Leidel et al., 2009). A prominent hypothesis of why U34 modifications become more critical during stress suggests that codons read via U34-modified tRNAs may be more frequent than average in stress response transcripts, decreasing their translation upon modification loss (Begley et al., 2007). However, this hypothesis has not been directly tested, and whether the absence of U34 modifications has any distinct consequences for translation during stress remains unknown. To define how U34 modifications maximize cellular fitness, we explored the consequences of their absence for codon translation dynamics in Saccharomyces cerevisiae and Caenorhabditis elegans. Using ribosome profiling (Ingolia et al., 2009), we found that tRNAs with mcm5s2U34 rely on modified U34 for efficient decoding of their cognate codons in vivo. Surprisingly, the codon-specific ribosome pausing we detected upon loss of U34 modifications in yeast was not augmented by environmental stresses. Instead, we discovered that slower decoding via hypomodified tRNAs elicits the aggregation of many essential proteins and profoundly impairs the ability of cells to balance protein homeostasis during stress. Our findings rationalize how loss of U34 modifications triggers cellular dysfunction and reveal a hitherto unappreciated role of codon translation rates for proteome integrity. (B) Codon-specific changes in A-site ribosome occupancy in S. cerevisiae strains with aberrant U34 modification in comparison to WT (mean ± SD; n = 3). Codons cognate for tRNAs with mcm5s2U34 (yellow), mcm5U34 (blue), and ncm5U34 (cyan) are boxed. Symbol size reflects the relative frequency of each codon within the A site in WT (small, 0.015). (C) Codon-specific changes in A-site ribosome occupancy upon U34 thiolation loss in C. elegans compared to WT (n = 2). Codons cognate for tRNAs with mcm5s2U34 are boxed in yellow; symbol size as in (B). See also Figures S1 and S2.

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RESULTS U34 Modifications Increase Translation Rates at a Subset of Cognate Codons in Yeast To establish the impact of U34 modifications on codon translation rates in vivo, we used ribosome profiling, which comprises deep sequencing of 30 nt mRNA fragments protected from nuclease digestion by bound ribosomes (Ingolia et al., 2009). This method provides a quantitative snapshot of ribosome occupancy along endogenous transcripts and allows the identification of codons in the ribosomal exit (E), peptidyl (P), and A sites (Ingolia et al., 2009; Stadler and Fire, 2011) (Figures S1A and S1B). As ribosome pausing typically increases the likelihood of capturing a footprint by sequencing, variations in codon-specific ribosome occupancy can be used to infer changes in translation rates (Ingolia et al., 2011; Li et al., 2012; Stadler and Fire, 2011). To stabilize translating ribosomes in a stage of the elongation cycle prior to peptide bond formation (Lareau et al., 2014) and to prevent runoff elongation during cell harvesting and ribosomal footprint isolation, cells were briefly treated with cycloheximide. We examined the contributions of mcm5/ncm5 and s2 moieties at U34 to translation by comparing codon-level ribosome occupancy in wild-type (WT) yeast to that of mutants with various U34 modification defects. Complete loss of U34 modifications is lethal in the W303 strain (Bjo¨rk et al., 2007), but we found that simultaneous knockout of Urm1 pathway components and Elp complex genes was compatible with cell viability in the S288c background. Thus, we performed ribosome profiling on ncs2Delp6D yeast (lacking all U34 modifications), as well as ncs2D (lacking 2-thiolation), and elp6D (lacking mcm5/ncm5 modifications and with lower 2-thiolation levels). Our analysis revealed strikingly distinct effects of U34 modification loss on codon occupancy. CAA and AAA triplets, read by the mcm5s2U34-containing tKUUU and tQUUG, were reproducibly enriched (20%–36%) within the ribosomal A site in all mutants (Figures 1B and S1C), suggesting they are translated more slowly when U34 is partially or fully unmodified. The increase in ribosome occupancy at these codons was larger and markedly more robust than previously reported for U34 thiolation-deficient strains (Zinshteyn and Gilbert, 2013). Occupancy at CAA and AAA was also consistently elevated when ribosome profiling was carried out in the absence of cycloheximide (Figure S1D), although the magnitude of the effect was smaller, likely due to translational runoff during sample preparation. There was little effect on ribosome occupancy at most other codons cognate for hypomodified tRNAs, including GAA, which is also decoded by a tRNA with mcm5s2U34 (Figure 1B). Curiously, a small but reproducible decrease in A-site ribosome occupancy was evident at AGA and GGA in elp6D and ncs2Delp6D cells (Figure 1B), indicating that these codons are translated faster when their cognate tRNAs (tRUCU and tGUCC) lack mcm5 at U34. Isoacceptors with U34 are unlikely to contribute to decoding via U:G wobble in yeast, evidenced by the minor effects of loss of U34 modifications on ribosome occupancy at G-ending codons (Figure 1B). Next, we asked whether U34 modifications mediate decoding specificity, reasoning that if aberrant U34 modification relaxes the specificity of codon binding, hypomodified tRNAs could compete for binding at near-cognate codons with mismatches 1608 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

at the third position, eliciting translational pausing. Such events could decrease decoding accuracy in split codon boxes, where two different amino acids are specified by a purine or a pyrimidine at the third position. Ribosome occupancy of AAC/U, CAC/U, or GAC/U codons was not altered in ncs2D yeast (Figure 1B), suggesting that U34 2-thiolation does not restrict the decoding specificity of tEUUC, tKUUU, and tQUUG. In contrast, the mcm5 group in U34 of tQUUG may be a prerequisite for accurate decoding in the Glu/His split codon box, as the A-site occupancy of CAC and CAU codons was reproducibly increased in elp6D and ncs2Delp6D cells (Figure 1B). Ribosome occupancy of AAC/U, GAC/U, and AGC/U codons was largely unaffected in elp6D and ncs2Delp6D cells, suggesting that a decrease in decoding accuracy is not a universal consequence of ablating mcm5U34. To assess if the global increase in ribosome occupancy at CAA and AAA in U34 modification-deficient yeast stems from slowed elongation at many instances of these codons, we compared the A-site ribosome occupancy at individual codons in wild-type and ncs2Delp6D yeast. We found that a greater proportion of CAA and AAA codons had high A-site occupancy in ncs2Delp6D cells (Figure S1E), indicative of widespread translational slowdown. By contrast, single-codon A-site occupancy at GAA and GCU was largely comparable in both strains, consistent with our global codon occupancy measurements (Figure 1B). We also asked whether codon occupancy in cells with unmodified U34 is modulated by the absence of known ribosome rescue factors. The Dom34-Hbs1 complex facilitates the dissociation of stalled ribosomes (Shoemaker and Green, 2012), and GTPBP2, a binding partner of the Dom34 homolog Pelota, was recently shown to resolve codon-specific translational stalling caused by depletion of a specific tRNA in the mouse brain (Ishimura et al., 2014). However, deletion of DOM34 or HBS1 in ncs2Delp6D yeast did not appreciably augment ribosome occupancy at CAA and AAA (Figure S1F) and had minimal phenotypic consequences (Figure S1G). Taken together, these findings argue against stalled ribosomes contributing significantly to the phenotypes of U34 modification mutants and suggest that increased codon occupancy in the mutant strains results from ribosome pausing, rather than from stalling. Overall, our data reveal a surprising heterogeneity in the outcomes of U34 hypomodifications for codon translation speed in yeast: while mcm5s2U34 in tKUUU and tQUUG is required for accurate and efficient translation elongation, U34 modifications play only minor functional roles in most other cytoplasmic tRNAs. Our observations are consistent with the phenotypic rescue of U34 modification defects by elevated levels of tKUUU and tQUUG, with a minor contribution of tEUUC (Bjo¨rk et al., 2007; Esberg et al., 2006; Leidel et al., 2009) (Figure S1H). Codon Translation Rates Are Augmented by U34 Modifications in C. elegans To determine whether U34 modifications modulate codon translation rates in a multicellular organism, we compared the codonspecific ribosome occupancies of wild-type C. elegans (N2) and tut-1(tm1297), a U34 2-thiolation-deficient strain (Chen et al., 2009a; Dewez et al., 2008; Leidel et al., 2009). The mutant displayed a robust 1.5- to 2.5-fold increase in A-site ribosome occupancy at AAA, CAA, and GAA (Figures 1C and S2). No

Figure 2. Codon-Specific Pausing in U34 Modification-Deficient Yeast Is Not Enhanced during Stress

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enrichment of near-cognate codons was detectable in the A site within ribosome footprints from tut-1(tm1297) worms, similarly to ncs2D yeast (Figure 1B). Therefore, the function of U34 2-thiolation in promoting efficient translation of cognate codons is conserved from yeast to metazoans. U34 Modifications Are Dispensable for Stress Responses in Yeast To test whether codon-specific ribosome pausing increases during stress, we exposed wild-type and ncs2D yeast to diamide or

(A) Experimental design to assess the effect of U34 modification loss during adaptation to stress in vivo. (B–E) Log2 fold changes in mRNA abundance and gene-level ribosome occupancy in WT and ncs2D cells exposed to diamide or rapamycin in comparison to WT grown in YPD (n = 3). Genes with statistically significant changes (Benjamini-corrected p < 0.01) for mRNA levels (orange), ribosome occupancy (gray), or both (red) are indicated. Pearson correlation coefficient (r) between ribosome occupancy and mRNA abundance changes is shown. (F) Hierarchically clustered heat map of ribosome occupancy data in (B)–(E). (G) Codon-specific changes in A-site ribosome occupancy in ncs2D yeast after diamide or rapamycin exposure (mean ± SD; n = 3). Data for ncs2D cells grown in YPD are from Figure 1B. (H) Codon-specific changes in A-site ribosome occupancy in elp6D yeast after rapamycin exposure (mean ± SD; n = 3). Data for elp6D cells grown in YPD are from Figure 1B. See also Figure S3 and Table S1.

rapamycin and subjected them to ribosome profiling, while monitoring changes in mRNA abundance (Figure 2A). Treatment with either drug induced a dramatic remodeling of gene expression in comparison to normal growth. Hundreds of genes displayed significant changes in transcript levels and ribosome occupancy that were remarkably concordant (Figures 2B–2E; Table S1), emphasizing the key role of mRNA abundance for regulating gene expression upon stress (Preiss et al., 2003). Gene expression signatures associated with the environmental stress response in yeast (Gasch et al., 2000) were prominent in both ncs2D and wildtype yeast. The induced genes comprised mediators of oxidative stress responses, protein folding and degradation, and respiration, whereas genes associated with energetically costly processes, such as ribosome biogenesis and translation, were profoundly repressed (Table S1; data not shown). Reduced translation in response to both compounds was also evident in a decreased polysome-to-monosome ratio (Figure S3A). Surprisingly, ncs2D cells were not overtly compromised in mounting a response to either treatment, since the extent and magnitude of changes in mRNA abundance and ribosome occupancy were similar in wild-type and the mutant (Figures 2B–2F). These data indicate that loss of U34 modifications does not impact the reprogramming of gene expression initiated in response to environmental stress. Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors 1609

As the composition of the transcript pool bound by ribosomes was dramatically different between rapidly growing and stressed cells but highly similar in wild-type and ncs2D yeast grown under the same conditions (Figure 2F), we could directly assess whether codon-specific translational pausing is magnified during stress. The codon-specific differences in ribosome occupancy in ncs2D exposed to diamide or rapamycin were comparable to those observed during normal growth (Figures 2G and S3B), showing that global demand for U34-modified tRNAs does not change upon environmental stress. We obtained analogous results when comparing A-site occupancy in elp6D yeast during normal growth or after rapamycin exposure (Figure 2H). The only discernible quantitative difference was a moderate increase of A-site occupancy at GAA during stress (Figures 2G and 2H); given the minor importance of tEUUC for phenotypic suppression (Esberg et al., 2006; Leidel et al., 2009) (Figure S1H), we consider this increase unlikely to be functionally relevant. Taken together, these data demonstrate that neither the extent of codon-specific ribosome pausing nor the ability to mount stress responses are altered in U34 modification-deficient cells. U34 Modification Defects Trigger Proteotoxic Stress in Yeast To explore alternative explanations for the diminished stress tolerance of yeast with aberrantly modified U34, we examined whether they display adaptive responses during normal growth. Indeed, when compared to wild-type, numerous genes in the mutants showed statistically significant changes both in mRNA abundance and gene-level ribosome occupancy (Figures 3A– 3C). These changes were highly concordant (Pearson r = 0.74– 0.85), indicating that reprogramming of gene expression in the mutants occurs predominantly via altered transcript levels. We noted a considerable overlap among the differentially expressed genes in ncs2D, elp6D, and ncs2Delp6D cells (Figures 3E and 3F), implying a common trigger for the cellular response to aberrant U34 modification. To characterize this response, we focused on genes in ncs2Delp6D yeast with significantly altered ribosome occupancy. The functional enrichment in downregulated genes was remarkably reminiscent of the core response to environmental stress in yeast: repression of genes related to protein synthesis (Figure 3F; Table S2) aimed at conserving energy (Gasch et al., 2000). Decreased abundance of proteins from these functional groups has previously been argued to occur without concomitant changes in transcript levels and to result from inefficient translation of codons for K, Q, and E in U34 modification-deficient yeast (Laxman et al., 2013; Rezgui et al., 2013). However, we found that decreased ribosome occupancy in ncs2Delp6D cells correlated strongly with a reduction in mRNA abundance (r = 0.85; Figure 3C; Table S2) and is thus unlikely due to defects in translation. Intriguingly, the genes induced in the ncs2Delp6D strain were strongly enriched for components of the protein degradation and refolding machinery (Figures 3G and 3H), suggesting that protein homeostasis may be perturbed. Genes encoding catalytic and regulatory subunits of the proteasome (PRE2, PRE3, PRE10, PUP1, PUP3, RPT1–RPT5, and others), heat shock proteins from the cytosolic disaggregation and refolding machinery 1610 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

(HSP104, HSP42, HSP82, SSA3, and SSA4), and membrane chaperones (HSP12 and HSP30) all had significantly higher mRNA levels and ribosome occupancy in ncs2Delp6D cells (Table S2). The induction of ubiquitin-proteasome system (UPS) genes in the mutant falls in the range observed upon acute proteotoxic insults, such as diamide exposure (2-fold; Table S1) (Gasch et al., 2000). Given the high intracellular abundance of UPS components, this increased expression is likely to incur substantial metabolic costs, prompting us to examine its significance for cellular fitness. Decreasing UPS levels via genetic ablation of the transcription factor Rpn4 (Xie and Varshavsky, 2001) profoundly delayed the growth of ncs2Delp6D (Figure 3I) and exacerbated the stress sensitivity of ncs2D, ncs6D, elp2D, and elp6D strains (Figures S4A and S4B), whereas it had little effect on growth in a wild-type background. We conclude that the ability of U34 modification-deficient strains to induce and maintain high UPS levels is critical for cellular fitness. Several other gene ontology (GO) terms were enriched among the transcripts induced in U34 modification-deficient cells, albeit to a much lower extent (Figure 3G). Among these were amino-acid biosynthesis genes and their transcription activator GCN4, which we found to be translationally upregulated in cells with hypomodified U34 (Table S2), consistent with previous observations (Zinshteyn and Gilbert, 2013). The absence of GCN4, however, did not overtly compromise the growth or stress sensitivity of yeast strains with hypomodified U34 (Figure S4C), demonstrating that activation of the Gcn4 pathway does not enhance cellular fitness in the absence of U34 modification. Endogenous Proteins Aggregate upon Loss of U34 Modifications in Yeast The prominent induction of the UPS, coupled with the upregulation of cytosolic chaperones, raises the intriguing possibility that loss of U34 modifications may perturb the conformational integrity of proteins. To test this, we performed a quantitative isolation of endogenous protein aggregates from exponentially growing wild-type and U34 modification-deficient cells (Koplin et al., 2010). Strikingly, we found that aberrant U34 modification elicits the aggregation of endogenous proteins, while aggregated species were virtually undetectable in the wild-type (Figure 4A; Table S3). The pattern of insoluble proteins was comparable in all mutants, but the extent of aggregation was most pronounced in the ncs2Delp6Dstrain. Since U34 modification defects lead to codon-specific translational pausing (Figure 1B), we considered that pausing might perturb cotranslational protein folding. Consistent with this possibility, we found that the absence of the ribosome-associated chaperones Ssb1 and Ssb2 aggravates the phenotypes of strains with hypomodified U34 (Figure S5A). Deletion of SSB1 and SSB2 is known to also elicit protein aggregation (Koplin et al., 2010; Willmund et al., 2013). Remarkably, we found that the extent of aggregation and the pattern of insoluble species in ncs2Delp6D and ssb1Dssb2D cells were very similar (Figure 4B). These data indicate that loss of U34 modification is detrimental for the solubility of many proteins that are destabilized also in the absence of cotranslationally acting chaperones. Using quantitative mass spectrometry, we identified 610 proteins

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Figure 3. Cells with U34 Modification Defects Exhibit Gene Expression Hallmarks of Proteotoxic Stress (A–C) Log2 fold changes (mutant/WT) in mRNA abundance and gene-level ribosome occupancy in U34 modification-deficient strains grown in YPD (n = 3). Color annotation is as in Figure 2B. (D) Hierarchically clustered heat map of ribosome occupancy data from (A)–(C). (E) Venn diagram of the overlap between significantly downregulated (Benjamini-corrected p < 0.01) genes in U34 modification-deficient strains. (F) GO term enrichment in genes significantly downregulated in ncs2D, elp6D, and ncs2Delp6D strains was summarized and visualized in semantic similaritybased scatter plots using REViGO. x axis: likeliness of meaning between GO terms; y axis: Benjamini-corrected p values of statistical significance for GO category enrichment; symbol size: frequency of GO term in database. (G) Venn diagram of the overlap between significantly upregulated genes. (H) GO term enrichment in genes upregulated in ncs2Delp6D calculated as in (F). (I) Growth defects of strains lacking Rpn4, which controls expression of the UPS via the proteasome-associated control element (PACE). See also Figure S4 and Table S2.

as significantly enriched in insoluble fractions from ncs2Delp6D, and 70% of these also aggregated in ssb1Dssb2D cells (Figure 4C). Approximately 40% of the aggregated proteins from both ncs2Delp6D and ssb1Dssb2D mutants are essential for yeast viability (Figure 4D). Importantly, increased aggregation propensity did not result from higher abundance of the insoluble proteins in the mutants when compared to wild-type (Figures S5B and S5C).

As the major effect of aberrant U34 modification on translation speed is pausing at CAA and AAA (Figure 1B), we asked whether the usage of these codons was disproportionally high in messages encoding aggregated proteins. Although we found that the fraction of glutamines encoded by CAA was increased in the ncs2Delp6D/ssb1Dssb2D overlap dataset (Figure S5D), the fact that these proteins aggregated in both strains suggests that the correlation with CAA codon content is spurious. Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors 1611

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Figure 4. Loss of U34 Modifications Elicits Protein Aggregation in Yeast and Nematodes (A) Detergent-insoluble protein aggregates isolated from WT and mutant strains were visualized by SDS-PAGE and Coomassie staining (left: total extracts; right: aggregates). (B) Protein aggregation patterns in the absence of ribosome-associated chaperones Ssb1/Ssb2 and upon loss of U34 modifications. (C) Quantitative mass spectrometry (MS) identification of proteins aggregating in ncs2Delp6D and ssb1Dssb2D yeast. (D) Fraction of essential proteins in aggregates from ncs2Delp6D and ssb1Dssb2D. (E and F) GO term enrichment in aggregates from ncs2Delp6D (D) and ssb1Dssb2D (E) cells calculated as in Figure 3F. (G) Q35-YFP aggregation in body-wall muscle cells is enhanced by loss of U34 modifications in C. elegans. Representative images of the head region from 1-day-old adult control and tut-1(tm1297), elpc-1(tm2149) animals are shown; Q35-YFP aggregates are indicated with arrows. Box plots depict the median aggregate number per animal (solid line), the 25% and 75% quartiles (box), and the 1.53 interquartile range (dashed lines) in data pooled from five independent experiments (n R 20 animals per experiment). ****p % 10 15, Wilcoxon test. See also Figures S5 and S6 and Table S3.

Moreover, lysines in aggregated proteins were marginally less likely to be encoded by AAA (Figure S5E). Given these results, and the large overlap between proteins aggregating upon such diverse conditions as U34 modification defects and chaperone loss (Figure 4C), we propose that many of the aggregated species abundant enough to be detected by mass spectrometry are metastable and thus highly vulnerable to perturbations in the cellular folding environment. Consistent with this idea, aggregates from both mutant strains contained many constituents of multisubunit assemblies (Figures 4E and 4F; Table S3), and the species we detected only in aggregates from ncs2Delp6D yeast were mostly additional subunits of complexes aggregating in both mutants (Table S3). Proteins that participate in large complexes have many interaction surfaces that can also cause aberrant associations (Pechmann et al., 2009), and their relative abundance exceeds their solubility in cells (Ciryam et al., 2013). Both properties have been suggested to predispose them to aggregation upon disruption of protein homeostasis. Indeed, during aging or upon proteotoxic challenge, constituents of the translational machinery and proteasomes aggregate in yeast, nematodes, and human cells (Ciryam et al., 2013; David et al., 2010; Kirstein-Miles et al., 2013; Koplin et al., 2010; Willmund et al., 2013). Thus, the widespread aggregation of endogenous metastable proteins upon loss of U34 modifications indicates an imbalance in protein homeostasis. Loss of U34 Modifications Impairs Protein Homeostasis in C. elegans To examine whether loss of U34 modifications disrupts protein homeostasis in C. elegans, we employed an aggregation-prone chimeric protein consisting of a stretch of 35 glutamine residues (Q35) fused to YFP. When expressed in body-wall muscle cells, Q35-YFP is soluble and diffusely localized throughout development, but begins to aggregate with the onset of adulthood (Morley et al., 2002). Aggregation of Q35-YFP is a sensitive sensor for protein folding defects in vivo, since it is enhanced in the presence of misfolded proteins (Gidalevitz et al., 2006). Quantification of Q35-YFP aggregate numbers in young Q35-YFP transgenic animals with unmodified U34 (tut-1(tm1297), elpc-1(tm2149); Chen et al., 2009a) revealed that aggregate burden was 2.5-fold higher in the mutant than in control animals (Figure 4G). Q35-YFP aggregation was similarly augmented after silencing of tut-1 by RNAi (Figure S5F). The aggregate burden is comparable to the one observed upon silencing of the ribosomebound chaperone NAC (3- to 4-fold; Kirstein-Miles et al., 2013). Moreover, U34 modification deficiency correlated with increased expression of cytosolic heat shock protein genes (Figure S5G) and shorter life span (Figure S5H) (Taylor and Dillin, 2011). Taken together, our results demonstrate that defects in U34 modification impair protein homeostasis in both nematodes and yeast. Overexpression of Hypomodified tEUUC, tKUUU, and tQUUG Restores Codon Translation Rates and Protein Homeostasis in U34 Modification-Deficient Cells To establish whether proteotoxic stress (Figure 3H) and aggregate accumulation (Figure 4A) in cells with aberrantly modified U34 stem from codon-specific translational pausing (Figure 1B),

we examined whether translation speed and protein homeostasis can be restored by tRNA overexpression. We used ribosome profiling to test whether ribosomal pausing at their cognate codons is mitigated in U34 modification-deficient yeast overexpressing tEUUC, tKUUU, and tQUUG. Indeed, while tRNA overexpression had no effect on growth or A-site occupancy in wildtype (Figures S6A and S6B), it markedly decreased occupancy at CAA and AAA in ncs2D and elp6D cells (Figures 5A and S6C). These data suggest that in U34 modification-deficient cells, ribosomes accept the hypomodified tRNAs less efficiently as a match to their cognate codons during decoding, leading to a perceived scarcity of these species when present at endogenous levels. To test this model further, we compared codon occupancy in ncs2Delp6D and wild-type cells after exposure to paromomycin, which increases near-cognate tRNA acceptance (Kramer et al., 2010). Pausing at CAA and AAA codons was mitigated by paromomycin (Figure 5B), providing further evidence that impaired codon-anticodon interactions within the ribosomal A site account for the slower codon translation rates in the absence of U34 modifications. Next, we asked whether restoration of codon translation rates is sufficient to improve protein homeostasis in yeast with hypomodified U34. Remarkably, we found that when intracellular levels of tEUUC, tKUUU, and tQUUG were augmented, the aggregate burden in ncs2D and elp6D cells decreased substantially (Figure 5C). Concomitantly, gene expression in the mutants reverted close to wild-type patterns upon tRNA overproduction (Figure 5D). The restoration of codon-specific translation speed therefore not only ameliorates protein aggregation but also obviates the need for increased protein quality control and repression of growth-related genes. Collectively, our findings indicate that protein homeostasis imbalance in U34 modification-deficient cells is elicited by slower decoding of CAA and AAA. Cells with Unmodified U34 Are Deficient in Resolving Stress-Induced Protein Aggregates Protein aggregation in cells with aberrantly modified U34 (Figure 4A) can reduce cellular fitness via loss of function of essential proteins (Figure 4D) and aggregate toxicity (Geiler-Samerotte et al., 2011). The chronic accumulation of protein aggregates is also known to compromise the capacity of cells to cope with misfolding triggered by acute proteotoxic stress (Hipp et al., 2014). We therefore asked whether impaired clearance of stress-induced protein aggregates might contribute to the stress sensitivity of cells with unmodified U34. For this, we monitored the subcellular localization of Hsp104, an aggregate-remodeling chaperone, in wild-type and ncs2Delp6D cells upon diamide exposure. Hsp104 is diffusely distributed throughout cells during normal growth, but upon proteotoxic insults, it localizes to distinct cytoplasmic foci containing spatially sequestered misfolded proteins (Escusa-Toret et al., 2013). GFP-tagged Hsp104 was uniformly distributed in exponentially growing wild-type and ncs2Delp6D cells. Within 1 hr of diamide exposure however >80% of cells from both strains harbored multiple Hsp104-GFP foci (Figures 6A and 6B), demonstrating that diamide is a potent inducer of protein aggregation in vivo. Whereas aggregates were rapidly eliminated from wild-type cells Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors 1613

A

C

Figure 5. Elevated Levels of Hypomodified tEUUC, tKUUU, and tQUUG Alleviate Ribosome Pausing and Relieve Proteotoxic Stress in U34 Modification-Deficient Yeast (A) A-site ribosome occupancy changes in ncs2D cells carrying an empty plasmid or overexpressing tEUUC, tKUUU, and tQUUG (tEKQ) in comparison to WT (mean ± SD; n = 3). (B) Effect of paromomycin on A-site codon occupancy in ncs2Delp6D yeast (mean ± SD; n = 3). Values for the ncs2Delp6D strain grown in YPD are derived from Figure 1B. (C) Effect of tEUUC, tKUUU, and tQUUG overexpression on protein aggregation in ncs2D and elp6D cells. (D) Heat map of log2 fold changes (compared to WT) for genes with statistically significant differences in ribosome occupancy in ncs2D (left panel) and elp6D (right panel) cells carrying an empty plasmid or overexpressing tEUUC, tKUUU, and tQUUG.

D compromises their ability to restore proteome integrity upon acute proteotoxic insults.

B DISCUSSION

upon transfer to fresh medium, they persisted significantly longer in ncs2Delp6D cells (Figure 6B). Aggregate elimination in the mutant was impaired to an even greater extent upon continuous diamide exposure: virtually all ncs2Delp6D cells still harbored Hsp104-GFP foci 4 hr after addition of the drug, as opposed to only 40% of wild-type cells (Figure 6C). Thus, the clearance of diamide-induced protein aggregates is severely delayed in cells with aberrantly modified U34, a result that correlates well with their increased sensitivity to this compound (Figures S1H and S3B). Given that diamide does not augment codon-specific ribosome pausing (Figure 2G), our findings suggest that chronic protein aggregation in U34 modification-deficient cells severely 1614 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

The non-uniform rate of peptide synthesis by ribosomes has been suggested to impact the folding of nascent polypeptides (Pechmann et al., 2013; Thanaraj and Argos, 1996). Experimental support for this model has been difficult to obtain due to the poorly defined determinants of codon translation rates in living cells. By probing the consequences of tRNA anticodon modification loss for translation in vivo, we show that codon-anticodon interactions are an evolutionarily conserved modulator of decoding speed in eukaryotes (Figures 1B and 1C). We further demonstrate that translational pausing at a small subset of codons severely disrupts protein homeostasis and triggers the widespread aggregation of endogenous proteins (Figures 3H and 4A). Thus, the stability of codon-anticodon interactions mediated by anticodon modifications emerges as a major determinant of codon translation rates and the integrity of the eukaryotic proteome. Modifications at U34 Accelerate Decoding In Vivo The magnitude of ribosome pausing triggered by U34 modification loss differs in yeast and nematodes (Figures 1B and 1C), since it is likely modulated by additional factors, such as the abundance of near-cognate tRNAs that can compete for A-site codon binding, mRNA topology, and the nature of the nascent chain. Pausing most likely results from inefficient decoding,

B proteotoxic stress protein aggregates Hsp104

WT diamide

Hsp104-GFP

untreated

100

Percentage of cells with Hsp104-GFP foci

A

Figure 6. Clearance of Stress-Induced Protein Aggregates Is Severely Compromised in Yeast with Unmodified U34

WT

80 60 40 20 0 0

30

60

90

120

150 180

210

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Time (min) diamide fresh medium

C

WT

diamide

Hsp104-GFP

untreated

Percentage of cells with Hsp104-GFP foci

100 80 60 40 20 0 0

30

60

90

120

150 180

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(A) Intracellular distribution of Hsp104-GFP in WT and ncs2Delp6D yeast grown in YPD (left) or treated with 1.5 mM diamide for 60 min (right). (B) Percentage of WT (black squares) and ncs2Delp6D cells (red diamonds) with Hsp104-GFPcontaining aggregates upon transient exposure to diamide. Exponentially growing cells were treated with 1.5 mM diamide for 60 min and returned to fresh YPD medium. At least 100 cells from three different fields were counted per time point (mean ± SD; n = 3). (C) Percentage of WT and ncs2Delp6D cells with Hsp104-GFP-containing aggregates in the continuous presence of 1.5 mM diamide. Quantification was performed as in (B). (D) Model for the molecular consequences of aberrant U34 modification.

240

Time (min) diamide

speed (Figures 5A, 5C, and 5D; S6C). Slower decoding may increase the probamisfolding metastable errors in bility of rare translation events, such as proteins proteins translation or folding CAA AAA misreading or frameshifting (Drummond EP protein quality and Wilke, 2008; Fluitt et al., 2007; Komar, control x proteotoxic 2009), or trigger cotranslational misfolding U34 stress refolding/ if a nascent chain is trapped in an off-target disaggregation SLOW DOWN folding intermediate (O’Brien et al., 2014). mcm5s2U34 imbalance in Thus, we expect that ubiquitous translaprotein homeostasis tional pausing at CAA and AAA (Figures protein increased aggregates aggregate burden 1B and S1E) will impose different functional lethality degradation costs on different proteins, depending on codon and amino acid context. As one example, lysines flanking aggregationsince it was relieved by elevated levels of hypomodified tRNAs prone amino acid stretches are thought to oppose aggregation (Figures 5A and S6C) and paromomycin (Figure 5B). Unexpect- (Rousseau et al., 2006), and slower translation of AAA codons edly, loss of mcm5U34 or ncm5U34 had only minor effects on de- that specify such ‘‘gatekeeper’’ residues might be especially coding speed in yeast, both during normal growth and during detrimental for protein folding. Future measurements of aminostress (Figures 2G and 2H). Since U34 modifications are dispens- acid misincorporation rates in endogenous proteins will clarify able for tRNA stability and charging (Johansson et al., 2008), our the frequency and type of translational errors in cells with undata suggest that 5-carbon moieties in many tRNAs have little modified U34. However, as such errors are likely to occur in function (Phizicky and Alfonzo, 2010). The importance of only a fraction of molecules from a protein species, it will be chalmcm5s2U34 for decoding efficiency may have been the driving lenging to define how the presence of a subset of erroneously force behind the evolution of enzymes modifying the 5-carbon synthesized polypeptides will impact the folding or function of of U34, and the minor functional consequences of these modifi- a given protein in the cell. cations for most other tRNAs with U34 might have decreased Regardless of its effect on individual proteins, ribosome the evolutionary pressure for higher substrate specificity. Alter- pausing at CAA and AAA codons (Figure 1B) clearly destabilizes natively, 5-carbon moieties at U34 could play a more prominent the conformation of many proteins with essential cellular funcrole in decoding in other eukaryotes; however, the full repertoire tions (Figures 4C and 4D). Given that the presence of a single of tRNA species with mcm5U34 or ncm5U34 is only known in aggregation-prone polypeptide is sufficient to promote aberrant budding yeast so far (Johansson et al., 2008). interactions between metastable endogenous proteins (Olzscha et al., 2011), it is conceivable that misfolding of only very few speDecoding Speed: A Safeguard against Protein cies can significantly reduce the capacity of cells with unmodiAggregation fied U34 to maintain proteome integrity. Misfolding would not To our surprise, a reduction in global translation rates of only two necessarily be correlated with a high frequency of CAA or AAA codons—CAA and AAA (Figure 1B)—led to widespread protein codons, as even a single codon substitution in a structurally aggregation and imbalanced protein homeostasis (Figures 3H, important region can substantially alter protein conformation 4A, and 4C), defects alleviated solely by restoring decoding (Kimchi-Sarfaty et al., 2007). Proteins or nascent chains that

D

x

Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors 1615

misfold upon translational pausing may occupy critical regulators of protein homeostasis and sequester them from conformationally dynamic species that depend on these regulators for proper folding. This model (Figure 6D) would account for the widespread aggregation of metastable proteins we observed in yeast with unmodified U34 (Figures 4C and 4D; Figure 6D), as well as the enhanced aggregation of the Q35-YFP reporter in U34 modification-deficient C. elegans (Figures 4G and S5F). Interestingly, loss of U34 modifications did not activate the endoplasmic reticulum unfolded protein response (UPR) in yeast, as judged by the lack of induction of HAC1, its key regulator, in the ncs2Delp6D strain (Table S2). UPR activation was not generally impaired in cells with hypomodified U34, because HAC1 ribosome occupancy was upregulated 10-fold in diamide-treated ncs2D cells, and mutant strains were not hypersensitive to inducers of ER stress like tunicamycin or dithiothreitol (data not shown). It is plausible that the integrity of ER proteins is less affected by changes in decoding speed of single codons during their synthesis due to the entirely different folding constraints imposed by co-translational translocation across the ER membrane. Sensing of co-translational misfolding may also be more efficient at the ER to avoid retro-translocating damaged lumenal proteins to the cytoplasm. Cellular Dysfunction upon Loss of U34 Modifications Is Caused by Disruption of Protein Homeostasis Previous models attempting to rationalize the stress sensitivity of yeast mutants with aberrantly modified U34 have proposed that frequent usage of codons read via U34-modified tRNAs in genes encoding stress response regulators may decrease their translational output in such mutants (Bauer et al., 2012; Begley et al., 2007). However, our ribosome-profiling data are inconsistent with a general increase in the translational demand for U34-modified tRNAs during stress, as ribosome pausing elicited by hypomodified tRNAs in yeast was similar in rapidly growing and stressed cells (Figures 2G and 2H). Moreover, U34 modification-deficient cells reprogrammed gene expression in response to both drugs in a manner similar to wild-type (Figure 2F). Taken together, these data argue against a specific role for U34-modified tRNAs in the regulation of stress responses. Instead, we propose that chronic proteotoxic stress causes the decreased fitness of cells lacking U34 modifications. The extensive aggregation of endogenous proteins and the induction of protein quality control pathways in these cells are consistent with this hypothesis (Figures 3H and 4C). Both events correlated with ribosomal pausing at CAA and AAA codons (Figures 5A, 5C, and 5D), suggesting they are triggered by translation and folding errors that arise from decreased codon translation rates. Notably, we found that the capacity of cells with unmodified U34 to re-establish protein homeostasis upon acute stress is strongly diminished (Figures 6B and 6C); this is a common consequence of chronic protein misfolding and probably occurs via overload of the chaperone and degradation machineries (Hipp et al., 2014). Failure of protein homeostasis is thus a major contributor to cellular dysfunction upon defects in U34 modification (Figure 6D). In conclusion, our study establishes a direct link between the kinetics of translation at individual codons and the conformational integrity of the proteome. The widespread protein aggre1616 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

gation elicited by slower decoding of only two codons highlights the extreme sensitivity of cells to local changes in the pace of translation elongation. Such changes can result from loss of U34 modifications via perturbed codon-anticodon interactions, as we described here or from the destabilization of single tRNA species (Ishimura et al., 2014), and both have been associated with neurodegeneration. As neurons are particularly vulnerable to the toxicity of protein aggregates, our findings provide a novel basis for understanding the pathologies that occur when tRNA modification or stability is compromised. EXPERIMENTAL PROCEDURES Strains and Growth Conditions Yeast strains were in the S288c BY4741 background and are listed in Table S4. Cultures were grown to mid-exponential phase (optical density 600 [OD600], 0.4–0.5) at 30 C, 200 revolutions per minute in yeast extract peptone dextrose (YPD) (Formedium). For tRNA overexpression, cells were transformed with the high-copy 2m-based plasmid pRS425 encoding a single copy each of tEUUC, tKUUU, and tQUUG (Leidel et al., 2009) and grown in SDLeu to maintain plasmid selection. To induce stress, 1.5 mM diamide or 12.5 nM rapamycin was added at OD600 = 0.4 for 30 min. C. elegans strains were in the N2 (Bristol) wild-type background and were handled using standard methods. The following transgenes and mutants were used: rmIs132[Punc-54::q35::yfp] (Morley et al., 2002), tut-1(tm1297) (Leidel et al., 2009), and elpc-1(tm2149) (Chen et al., 2009a). Ribosome Profiling and RNA-Seq Yeast or L4-stage nematodes were pulverized under cryogenic conditions in a freezer mill (Spex) and RNase I-treated extracts were resolved by sucrose gradient centrifugation. Ribosome-protected footprints were purified from monosome fractions by size selection and gel extraction. Sequencing libraries were prepared essentially as described (Ingolia et al., 2012; Stadler and Fire, 2011). Codon occupancy was calculated similarly to Stadler and Fire (2011). Differential expression was tested by DESeq, and GO term enrichment was summarized and visualized with REViGO. Additional details are available in the Supplemental Experimental Procedures. Analysis of Endogenous Protein Aggregates Aggregated proteins were isolated as in Koplin et al. (2010), resolved by SDS-PAGE, and visualized with Coomassie staining. Proteins enriched in aggregate fractions were identified by label-free quantitative mass spectrometry. Wild-type and ncs2Delp6D cells expressing Hsp104-GFP from the HSP104 chromosomal locus were grown to exponential phase in YPD, treated as indicated, fixed in 3.7% formaldehyde, and imaged on a Zeiss Axio Imager 2 microscope. ACCESSION NUMBERS Sequencing data are available at the Gene Expression Omnibus (accession number GSE67387). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, and four tables and can be found with this article online at http:// dx.doi.org/10.1016/j.cell.2015.05.022. ACKNOWLEDGMENTS We thank K. Buhne for technical support, S. Mitani, S. Tuck, P. Go¨nczy, and E. Liebau for reagents, H. Drexler and A. Nolte for mass spectrometry, F. Konert and C. Gra¨f for sequencing, D. Eccles, J.M. Vaquerizas, B. Habermann, R. Luna, and M. Delattre for advice, A. Bertolotti for insightful suggestions, and A.J. te Velthuis, S. Starck, P. Go¨nczy, G. Rabut, K. Bartscherer, and

E.J. Snijder for comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by the Max Planck Society and grants from the North Rhine-Westphalian Ministry for Innovation, Science and Research (314-40001009), and the European Research Council (ERC-2012-StG 310489-tRNAmodi) (to S.A.L.); D.D.N. received an EMBO Long-Term Fellowship (ALTF1291-2010). Received: October 21, 2014 Revised: February 28, 2015 Accepted: April 10, 2015 Published: June 4, 2015

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Supplemental Figures

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Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors S1

Figure S1. Effects of U34 Modification Loss on Ribosome Occupancy at Codons Flanking the A Site In Vivo, Related to Figure 1 (A) Cumulative coverage of 50 nucleotides from ribosome footprint reads mapping near start codons across all transcripts in S. cerevisiae (left) and C. elegans (right). Reads of length between 29 and 31 nucleotides (nt) mapped without mismatches are shown. The peak located 12-13 nt upstream of start sites is inferred to represent ribosomes poised for translation initiation that contain the AUG codon in their P-site. (B) Approach for determining codon representation in tRNA-binding sites within ribosome footprints inferred from (A). (C) Codon occupancy within P, E, and +1 sites in yeast with U34 modification defects compared to WT (mean ± SD; n = 3). (D) Codon-specific changes in A-site ribosome occupancy in ncs2D cells compared to WT when cycloheximide (CHX) was omitted from all steps of the ribosome profiling protocol (mean ± SD; n = 3). Data from CHX-treated ncs2D cells is from Figure 1B. (E) Cumulative distribution of A-site ribosome occupancy at individual AAA, CAA, GAA, and GCU codons in WT and ncs2Delp6D yeast. To calculate single-codon occupancy, data from three biological replicates were pooled, and the number of A-site reads at a particular codon was normalized to the average per-codon A-site read density in the ORF containing it (p values are from a one-sample Kolmogorov–Smirnov test). (F) Codon-specific changes in A-site ribosome occupancy in S. cerevisiae strains lacking U34 modifications and ribosome rescue factors (mean ± SD; n = 3). Note that the elevated occupancy at codons such as CCG and CGC in dom34Dncs2Delp6D and hbs1Dncs2Delp6D cells is likely caused by additive effects, as occupancy at these codons is increased in hbs1D yeast. (G) Exponentially growing cultures from the indicated strains were serially diluted and spotted on medium without additives (YPD) or containing 1.2 mM diamide or 1.9 nM rapamycin. Plates were imaged after 2 days of incubation at 30 C. (H) Cultures from the indicated strains carrying an empty vector or overexpressing isoacceptors for E, K, and Q with U34 (tEUUC, tKUUU, and tQUUG) or C34 (tECUC, tKCUU, and tQCUG) were grown to exponential phase, serially diluted, and spotted on the indicated plates. Images were taken after 2 days of incubation at 30 C.

S2 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

Figure S2. Codon Occupancy within P, E, and +1 Sites in U34 Thiolation-Deficient tut-1(tm1297) Nematodes Compared to WT, n = 2, Related to Figure 1 Symbol size as in Figure 1C.

Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors S3

Figure S3. Effects of Diamide and Rapamycin on Global Translation and Codon Occupancy, Related to Figure 2 (A) Polysome profiles of WT and ncs2D cells grown in YPD or after treatment with diamide or rapamycin. Extracts from samples subjected to ribosome profiling in Figures 1 and 2 were analyzed. The polysome/monosome ratio (P/M) was calculated by quantification of integrated peak areas. (B) Codon occupancy within P, E, and +1 sites in ncs2D cells exposed to diamide or rapamycin (mean ± SD; n = 3). Values for ncs2D grown in YPD are derived from Figure S1C.

S4 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

A

B

C

Figure S4. Phenotypes of U34 Modification-Deficient Yeast Strains Lacking RPN4 or GCN4, Related to Figure 3 (A) RPN4 deletion significantly increases the doubling time of ncs2D and elp6D in YPD (mean ± SD; n = 3, **:p % 0.01 using an unpaired two-tailed Student’s t test). (B and C) Cultures from the indicated strains were grown to exponential phase, serially diluted, and spotted on the indicated plates. Imaging was performed after 3 days of incubation at 30 C.

Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors S5

Figure S5. Loss of U34 Modifications Elicits Protein Aggregation in Yeast and Nematodes, Related to Figure 4 (A) Exponentially growing cultures from the indicated strains were serially diluted and spotted on the indicated plates. Imaging was performed after 3 days of incubation at 30 C. (B and C) Comparison of log2 fold enrichment of protein species in aggregates versus log2 fold changes in abundance in total protein for ncs2Delp6D (B) and ssb1Dssb2D yeast (C). Pearson correlation coefficient (r) is indicated. (D and E) Analysis of the fraction of glutamines encoded by CAA (D) and lysines encoded by AAA (E) in proteins aggregating only in ncs2Delp6D, ssb1Dssb2D, or in both mutants (overlap) compared to all yeast verified and uncharacterized ORFs. Boxplots depict the median (solid line), the 25% and 75% quartiles (box) and 1.5 x the interquartile range (dashed lines). Statistical significance was defined as p % 0.01 from a Wilcoxon test. **p % 106, ***p % 1012 (F) The number of Q35-YFP aggregates in muscle cells of one-day-old nematodes is increased by knockdown of tut-1 by RNAi. Animals were fed bacteria carrying an empty L4440 vector (control) or dsRNA targeting tut-1 throughout development. Data from three independent experiments (n = 50 animals) were pooled. Solid lines indicate the median number of aggregates, boxes delimit 25% and 75% quartiles, and dashed lines show 1.5 x the interquartile range. **p % 106, Wilcoxon test. (G) Fold changes in abundance of transcripts encoding cytosolic heat shock response genes (hsp-16.41, hsp-70) and chaperones resident in mitochondria (hsp-6, hsp-60) and the ER (hsp-3, hsp-4) in adult tut-1(tm1297), elpc-1(tm2149) nematodes compared to age-matched wild-type N2 animals. Data from two biological replicates was analyzed by DESeq and the cut-off for statistical significance was defined as a Benjamini-corrected p value of less than 0.05 (ns = not significant). (H) Kaplan-Meier survival curves of wild-type N2, tut-1(tm1297), and tut-1(tm1297), elpc-1(tm2149) animals (N2 median lifespan = 16 days versus tut-1(tm1297) median lifespan = 13 days, p < 0.0001, and versus tut-1(tm1297), elpc-1(tm2149) median lifespan = 9 days, p < 0.0001, Mantel-Cox log-rank test).

S6 Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors

Figure S6. Effect of tRNA Overexpression on Codon Occupancy, Related to Figure 4 (A) Doubling time of cultures from the indicated strains carrying an empty vector or overexpressing tEUUC, tKUUU, and tQUUG (+tEKQ) (mean ± SD; n = 3; **: p % 0.01 from an unpaired two-tailed Student’s t test; n.s. denotes not significant). (B and C) Effect of overexpression of tEUUC, tKUUU and tQUUG on A-site codon occupancy in WT (B) and elp6D (C). Note that the least used codons in yeast (CGA, CGG) exhibit larger fluctuations in the elp6D data sets due to lower read depth.

Cell 161, 1606–1618, June 18, 2015 ª2015 The Authors S7

Cell Supplemental Information

Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity Danny D. Nedialkova and Sebastian A. Leidel

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Yeast and Nematode Strains All yeast strains (Table S4) were in the S288c BY4741 background (Winzeler et al, 1999). Single-gene knockout strains were generated by sporulation of strains from the BY4743 heterozygous diploid gene deletion collection (Thermo Scientific) and tetrad dissection. Gene disruption cassettes conferring kanamycin resistance were generally exchanged with auxotrophic markers to circumvent cross-resistance to paromomycin. Double- and tripleknockout strains were created by mating and sporulation. Gene knockouts were verified by the absence of mRNA and ribosome footprint reads from targeted genes and/or PCR. C. elegans strains were in the Bristol N2 wild-type background (Brenner, 1974). Culture Conditions for Stress Treatment in Yeast Transcript levels change only transiently following environmental perturbations in yeast and the magnitude of abundance change is proportional to the severity of the stress (Gasch et al., 2000). Therefore, to maximally capture the translation of response transcripts upon exposure to diamide or rapamycin, we chose a 30-min treatment, which corresponds to the peak of transcript abundance changes (Gasch et al., 2000; Hardwick et al., 1999). The amount of each drug was titrated to fully preserve cell viability at the time of analyses and to elicit similar growth delays in cultures of wild-type yeast at later time points. Based on this analysis, 1.5 mM diamide and 12.5 nM rapamycin were selected for subsequent experiments. Paromomycin was added to exponentially growing cultures at 1 mg/ml for 30 min. Ribosome Profiling of Yeast and Nematodes Elongating ribosomes were stabilized by a brief (60-sec) treatment of yeast cultures with 100 µg/ml CHX, and cells were harvested by rapid vacuum filtration (~45 sec) and flashfreezing. Yeast or L4 stage nematodes were pulverized under cryogenic conditions in 20 mM Tris-HCl (pH=7.5), 100 mM NaCl, 10 mM MgCl 2 , 1% Triton X100, 0.5 mM DTT, 100 µg/ml CHX at 5 cps in a SPEX 6750 Freezer/Mill (SPEX SamplePrep). Lysates were clarified by

centrifugation at 4°C, 3,000 g for 3 min followed by an additional centrifugation step at 4°C, 10,000 g for 5 min. Extracts from yeast or nematodes were treated with 11.25 U or 7.5 U RNase I (Ambion) per OD 260 unit of extract for 60 min at 22°C with continuous mixing. After addition of 100 U SUPERase In (Ambion), ribosomes were resolved on 7%-47% sucrose density gradients in 50 mM Tris-HCl (pH=7.5), 50 mM NH 4 Cl, 12 mM MgCl 2 , 0.5 mM DTT, 100 µg/ml CHX for 3 h at 35,000 rpm, 4°C in a TH-641 rotor (Thermo Scientific). Gradients were fractionated by upward displacement at 0.75 ml/min with continuous monitoring of OD 254 values. Fractions were collected at 32-sec intervals, and SDS was immediately added to 1% in monosome fractions, which were flash-frozen and stored at -80°C. RNA was isolated from monosome samples by the hot acid phenol method. Ribosome footprints were purified by separation of monosome RNA on 15% polyacrylamide, 8M urea, 1×TBE gels, staining with SYBR Gold (Life Technologies) and excision of bands between RNA oligonucleotide markers of 28 nt and 32 nt (yeast) or 28 nt and 30 nt (nematode). Total RNA Extraction from Yeast and Poly(A) RNA Selection A fraction of yeast cultures used for ribosome profiling was kept for total RNA extraction. Cells collected by rapid vacuum filtration were resuspended in ice-cold 50 mM NaOAc (pH=5.5), 10 mM EDTA, followed by addition of SDS to 1% and homogenization in a Precellys 24 bead beater (Bertin Technologies) at power setting 6.5 for two cycles of 20 sec. Lysates were clarified by centrifugation at 4°C, 10,000 g for 5 min, treated with 100 µg/ml Proteinase K for 20 min at 60°C, and total RNA was extracted with acid phenol. One hundred µg of RNA were treated with TURBO DNase (Ambion) and poly(A) RNA was purified by two rounds of selection with Poly(A)Purist MAG Kit (Ambion) according to the manufacturer’s instructions. Purified RNA was fragmented by alkaline hydrolysis in 50 mM sodium bicarbonate (pH=9.2), 1 mM EDTA for 20 min at 95°C. Fragmented RNA was purified by ethanol precipitation and fragments of 50-80 nt were recovered from 15% polyacrylamide, 8M urea, 1×TBE gels stained with SYBR Gold.

Total RNA Extraction from C. elegans Age-synchronized wild-type N2 and tut-1(tm1297), elpc-1(tm2149) mutant animals grown at 20°C were collected by filtration at day one of adulthood, resuspended in a fourfold excess of Tri Reagent (Sigma Aldrich), and flash-frozen. After six cycles of thawing in a 42°C waterbath and freezing in liquid nitrogen, total RNA was extracted using standard protocols and treated with TURBO DNase. Generation of Deep Sequencing Libraries Sequencing libraries from ribosome-protected footprints or randomly fragmented poly(A)selected RNA were generated essentially as described in (Ingolia et al., 2012). 3’-adapter ligation was performed for 4 h at 22°C in a reaction containing 200 U of T4 RNA ligase 2 (truncated, NEB), 25% PEG 8000, and 10 U SUPERase In. Because rRNA contaminants in footprint libraries frequently originate from A-rich sequences (Brar et al., 2012; Guydosh and Green, 2014), we chose not to subtract those by antisense oligonucleotide hybridization to avoid unspecific depletion of footprints that could potentially skew the occupancy measurements of A-rich codons. Sequencing libraries of poly(A)-enriched RNA from adult N2 and tut-1(tm1297), elpc-1(tm2149) nematodes were generated with the TruSeq RNA Sample Prep Kit v2 (Illumina). Sequencing Data Analysis Sequencing data from ribosome footprints was pre-processed by trimming linker sequences from the 3’ end of reads and alignment to rRNA genes with Bowtie 1.0.0 (Langmead et al., 2009). Non-rRNA reads longer than 25 nt were mapped with Bowtie to reference sets consisting of ORFs in the sacCer3 genome annotated as verified or uncharacterized in the Saccharomyces Genome Database on Jan 07, 2013 (5,906 sequences) or UCSC canonical transcripts (Hsu et al., 2006) of the C. elegans WS190 (ce6) genome (20,051 sequences). In both cases, 18 nt of 5’ UTR and 3’ UTR sequences of each ORF were included to allow alignment of ribosome footprints from initiating and terminating ribosomes. One mismatch in the seed sequence was allowed during mapping (except for C. elegans mRNA-Seq data,

where two mismatches were allowed). Only reads with unique matches to the reference were retained for further analysis. The majority of footprint reads aligning to coding sequences had a length between 29 and 31 nt, within the range of previous observations (Ingolia et al., 2009; 2011). The total reads in each ribosome footprint library were 10 – 36 million (yeast) and 23 – 44 million (nematode). Of those, 5 – 20 million and 2 - 14 million mapped uniquely to coding regions, with the exception of libraries from paromomycin-treated yeast, which had lower depth (1 – 2 million uniquely mapping reads). For global codon occupancy measurements, only reads with a perfect match to the reference were used. The offset of P site codons from the 5’ ends of footprints was inferred by examining the cumulative distribution of 29-31 nt reads aligning at annotated CDS start codons, which are positioned in the P site of initiating ribosomes (Ingolia et al., 2009; Jackson et al., 2010). In agreement with previous reports (Ingolia et al., 2009; Stadler and Fire, 2011), we found that footprint coverage begins abruptly 12 to 13 nt upstream of start codons in both S. cerevisiae and C. elegans (Figure S1A). For high-precision assignment of P site codons, we therefore selected reads with a 5’-nt in the 0 and -1 frames of canonical coding sequences, discarded the first nucleotide of reads beginning in the -1 frame, and used an offset of 12 nt from the 5’ end of each read to infer the P site codon (positions 13-15, Figure S1B). Since ribosome density in the beginning of ORFs can be significantly affected by growth conditions, as well as ongoing translation initiation or ribosomal run-off during sample preparation (Gerashchenko and Gladyshev, 2015; Ingolia et al., 2009; 2011), we excluded footprint reads mapping within the first 15 codons of coding sequences from our analyses. The frequency of each codon in different sites within footprints was then calculated. To obtain the basal occurrence of each codon, we computed its average frequency in the three non-tRNA binding positions downstream of the A site (+1 to +3, Figure S1B). These positions have not yet been translated and codon identity, therefore, should have negligible effects on ribosome occupancy. Codon representation in E, P, and A sites was then computed by dividing the frequency of a codon in each of these sites by its basal occurrence, an approach similar to the one described in (Stadler and Fire, 2011). Using this metric, we were able to detect known ribosome pausing

events, such as those associated with the presence of proline codons within the P site in both mouse ES cells and S. cerevisiae (Ingolia et al., 2011; Zinshteyn and Gilbert, 2013), as well as when codons translated via G:U wobble occupied the P site in C. elegans (Stadler and Fire, 2011) (data not shown). For comparing A-site codon occupancy at individual positions, the single-codon occupancy metric described in (Zinshteyn and Gilbert, 2013) was used. Briefly, the A-site codon in reads used for global codon occupancy measurements was assigned according to the criteria outlined above, and read counts at a given codon were normalized to the average per-codon read density in the ORF containing it. The first 15 codons of each ORF and codons with no reads were excluded from this analysis. For gene-level measurements, differential ribosome occupancy (excluding the first 15 codons) and mRNA abundance were tested with DESeq, which uses a model based on the negative binomial distribution (Anders and Huber, 2010). Statistical significance was defined by a Benjamini-corrected p-value of less than 0.01, and low coverage genes were filtered out by requiring a mean value of normalized reads between two samples being compared of at least 50. Gene Ontology (GO) term enrichment was analyzed with the functional annotation tool of the DAVID bioinformatics web server (http://david.abcc.ncifcrf.gov/). GO terms with a Benjamini-corrected p-value of less than 0.01 were summarized and visualized in semantic similarity-based scatterplots using the REViGO web server (http://revigo.irb.hr/, Supek et al., 2011). Isolation of Endogenous Protein Aggregates from Yeast Protein aggregates were isolated as in (Koplin et al., 2010). Wild-type and knockout yeast strains were grown to mid-log phase (OD 600 ~0.5) at 30°C and cells from 100 ml of culture were harvested by rapid vacuum filtration and flash-frozen. After resuspension in extraction buffer (20 mM NaPi pH=6.8, 1 mM EDTA, 10 mM DTT, 0.1% Tween 20) containing a protease inhibitor cocktail (0.5 mM AEBSF, 10 µg/ml aprotinin, 0.5 mg/ml benzamidine, 20 µM leupeptin, 5 µM pepstatin A), 60 U of zymolyase T20 (Zymo Research) were added, and the cell

suspension was incubated at 22°C for 20 min with continuous mixing. Extracts were chilled on ice and sonicated eight times at level 4, duty cycle 50% with a Branson tip sonicator. Cell debris was sedimented by centrifugation at 200 g, 4°C for 20 min and protein concentration in supernatants was determined with the Bio-Rad Protein Assay. Protein concentration was equalized across samples and aggregates were pelleted from equal amounts of total protein by centrifugation at 16,000 g, 4°C for 20 min. Pellets were resuspended in washing buffer (20 mM NaPi pH=6.8, 2% NP-40, protease inhibitor cocktail) by sonicating six times at level 4, duty cycle 50%, and sedimented again at 16,000 g, 4°C for 20 min. After a second wash in buffer B, aggregates were resuspended in detergent-free washing buffer by sonicating 4 times at level 2, duty cycle 65%. Following sedimentation, pellets were dissolved in Laemmli sample buffer with 100 mM DTT and 8M urea by boiling at 95°C for 10 min. Total protein extracts and aggregates were separated on 4 -12% NuPAGE Bis-Tris gels (Life Technologies) in MES-SDS running buffer and gels were stained with Colloidal Blue Staining Kit (Life Technologies). Quantitative Protein Mass Spectrometry Isolated aggregates and matched total protein samples were size-separated by SDS-PAGE and subjected to in-gel trypsin digestion (Shevchenko et al., 2007). Extracted peptides were desalted using Empore-C18 StageTips (Rappsilber et al., 2003) and stored at 4°C until further use. Prior to LC-MS/MS, peptides were eluted using 2 x 20 µl of 80% ACN, 0.5% acetic acid, dried in an Eppendorf concentrator to a volume of about 2 µl, and resuspended in 10 µl Buffer A (0.5% acetic acid). 6 µl of this peptide solution were subsequently analyzed by nanoscale reversed-phase chromatography using a EASY nLC 1000 UHPLC on a PepMap C18 EASYSpray™ column (15 cm x 75 µm ID, 3 µm particle diameter; Thermo Scientific; column temperature set to 45°C) that was online coupled via an EASY-Spray™ ESI source (Thermo Scientific) to a Q Exactive mass spectrometer (Thermo Scientific). Peptides were separated at a flow rate of 250 nl/min using a multistep gradient (3 – 25% B in 60 min; 25 – 32% B in 10 min; 32 – 95% B in 5 min; hold at 95% B for 5 min) before re-equilibration at starting conditions. The mass spectrometer was operated in data-dependent mode, acquiring full scan spectra at a

resolution of 70.000 and an AGC target value of 3e6 (scan range 300 - 1750 m/z). The ten most intense ions were chosen for higher energy collisional dissociation (HCD) with a resolution of 17.500 at m/z 400 and a target value of 1e6. Dynamic exclusion was allowed and set to 20 sec, uncharged as well as singly charged compounds were excluded from the analysis. Data were recorded with Xcalibur (Thermo Scientific). Raw MS files were processed using the MaxQuant computional platform (version 1.4.1.2) (Cox and Mann, 2008). Identification of peptides and proteins was enabled by the built-in Andromeda search engine by querying the concatenated forward and reverse yeast Uniprot database, release 2013-02, including common contaminants. The allowed initial mass deviation was set to 7 ppm and 20 ppm in the search for precursor and fragment ions, respectively. Trypsin with full enzyme specificity and only peptides with a minimum length of 7 amino acids were selected, and a maximum of two missed cleavages was allowed. Carbamidomethylation (Cys) was set as fixed modification, while oxidation (Met) and Nacetylation were defined as variable modifications. Protein and peptide identification was performed at a false discovery rate (FDR) of 1%. Relative label free quantification was based on the measurements of three independent biological replicates for each strain analyzed by MaxQuant with the ‘match between runs’ option turned on. Data processing and annotation were performed using the Perseus module of MaxQuant (version 1.4.0.6). Reverse and contaminant hits, as well as those identified by a modified site only, were filtered out. At least one unique peptide was required for protein identification. Intensity values were logarithmized and missing values were replaced by imputation, simulating signals of low abundant proteins within the distribution of measured values using a width of 0.3 SD and a downshift of 1.8 SD. Statistical significance was determined by two-sample t tests with a permutation-based FDR cutoff of 5%. For aggregate samples, proteins identified in ncs2Δelp6Δ and ssb1Δssb2Δ samples were considered aggregated only if enriched at least 2-fold over wild type. A small number of paralogous proteins that passed these criteria differ by as little as one residue and could not be

distinguished in our MS data despite high sequence coverage. In such cases, we included both paralogs in the set of aggregated proteins and denoted their enrichment value as “undefined”. C. elegans RNAi and Lifespan Analysis For RNAi experiments, E. coli HT115(DE3) carrying the double-stranded-RNA-expressing vector L4440 were seeded onto NGM plates containing 2mM IPTG and 50 µg/ml carbenicillin, and dsRNA expression was induced overnight at room temperature. Nematodes expressing a CAG-encoded polyQ stretch fused to YFP (Q35-YFP, Morley et al., 2002) were synchronized by egg lay on RNAi plates and grown at 20°C. Animals were transferred to fresh plates every 24 to 48 h and Q35-YFP aggregates in body wall muscle cells was imaged in one-day-old adults (four days after egg lay) on a Zeiss Axio Imager 2 microscope. RNAi treatment was unknown to the researcher during aggregate quantification. Lifespan analysis was performed at 20°C on E. coli OP50. Nematodes were agesynchronized by egg lay and the young adult stage was defined as day 0. One hundred animals were used per condition and were scored for viability every day or every second day. Animals that had crawled off the plate or undergone internal hatching were censored. Statistical analysis was performed with GraphPad Prism using the log-rank (Mantel–Cox) method.

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