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. 2015 Jun 18;161(7):1606-18.
doi: 10.1016/j.cell.2015.05.022. Epub 2015 Jun 4.

Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

Danny D Nedialkova  1 , Sebastian A Leidel  2
Affiliations

Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

Danny D Nedialkova et al. Cell. .

Abstract

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.

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Figures

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Graphical abstract
Figure 1
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. (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, <0.005; medium, 0.005–0.015; large, >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.
Figure 2
Figure 2
Codon-Specific Pausing in U34 Modification-Deficient Yeast Is Not Enhanced during Stress (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 ncs2Δ 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 ncs2Δ yeast after diamide or rapamycin exposure (mean ± SD; n = 3). Data for ncs2Δ cells grown in YPD are from Figure 1B. (H) Codon-specific changes in A-site ribosome occupancy in elp6Δ yeast after rapamycin exposure (mean ± SD; n = 3). Data for elp6Δ cells grown in YPD are from Figure 1B. See also Figure S3 and Table S1.
Figure 3
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 ncs2Δ, elp6Δ, and ncs2Δelp6Δ strains was summarized and visualized in semantic similarity-based 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 ncs2Δelp6Δ 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.
Figure 4
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 ncs2Δelp6Δ and ssb1Δssb2Δ yeast. (D) Fraction of essential proteins in aggregates from ncs2Δelp6Δ and ssb1Δssb2Δ. (E and F) GO term enrichment in aggregates from ncs2Δelp6Δ (D) and ssb1Δssb2Δ (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 1.5× interquartile range (dashed lines) in data pooled from five independent experiments (n ≥ 20 animals per experiment). ∗∗∗∗p ≤ 10−15, Wilcoxon test. See also Figures S5 and S6 and Table S3.
Figure 5
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 ncs2Δ 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 ncs2Δelp6Δ yeast (mean ± SD; n = 3). Values for the ncs2Δelp6Δ strain grown in YPD are derived from Figure 1B. (C) Effect of tEUUC, tKUUU, and tQUUG overexpression on protein aggregation in ncs2Δ and elp6Δ cells. (D) Heat map of log2 fold changes (compared to WT) for genes with statistically significant differences in ribosome occupancy in ncs2Δ (left panel) and elp6Δ (right panel) cells carrying an empty plasmid or overexpressing tEUUC, tKUUU, and tQUUG.
Figure 6
Figure 6
Clearance of Stress-Induced Protein Aggregates Is Severely Compromised in Yeast with Unmodified U34 (A) Intracellular distribution of Hsp104-GFP in WT and ncs2Δelp6Δ yeast grown in YPD (left) or treated with 1.5 mM diamide for 60 min (right). (B) Percentage of WT (black squares) and ncs2Δelp6Δ cells (red diamonds) with Hsp104-GFP-containing 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 ncs2Δelp6Δ 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.
Figure 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 5′ 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 ncs2Δ 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 ncs2Δ cells is from Figure 1B. (E) Cumulative distribution of A-site ribosome occupancy at individual AAA, CAA, GAA, and GCU codons in WT and ncs2Δelp6Δ 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 dom34Δncs2Δelp6Δ and hbs1Δncs2Δelp6Δ cells is likely caused by additive effects, as occupancy at these codons is increased in hbs1Δ 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.
Figure S2
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.
Figure S3
Figure S3
Effects of Diamide and Rapamycin on Global Translation and Codon Occupancy, Related to Figure 2 (A) Polysome profiles of WT and ncs2Δ 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 ncs2Δ cells exposed to diamide or rapamycin (mean ± SD; n = 3). Values for ncs2Δ grown in YPD are derived from Figure S1C.
Figure S4
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 ncs2Δ and elp6Δ 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.
Figure 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 ncs2Δelp6Δ (B) and ssb1Δssb2Δ 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 ncs2Δelp6Δ, ssb1Δssb2Δ, 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 ≤ 10−6, ∗∗∗p ≤ 10−12 (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 ≤ 10−6, 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).
Figure S6
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 elp6Δ (C). Note that the least used codons in yeast (CGA, CGG) exhibit larger fluctuations in the elp6Δ data sets due to lower read depth.
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