tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding

Abstract

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

Keywords: SILAC; systems biology; translation regulation.

Fig. 1.

General translation is unaffected by lack of URM1. (A) Density plot of log₂(WT/urm1∆) protein abundance ratios from a single SILAC-based proteomics experiment. (B) Wild-type and urm1∆ cells were pulsed with [³⁵S]Met and [³⁵S]Cys in the presence (+) or absence (−) of cycloheximide (CHX). [³⁵S] incorporation into proteins was quantified by liquid scintillation counting. Counts per minute were normalized to the wild-type value. Data show mean ± SEM of three independent experiments. (C) Quantification of the polysome profiles from wild-type, urm1∆, and uba4∆ cells separated on a 6–45% sucrose gradient showing the distribution of 40S, 60S, and 80S particles and polysomes as percentage of total ribosomes from the average of three independent experiments. See also Fig. S1.

Fig. 2.

URM1 is important for efficient expression of a subset of AAA-, CAA-, AAG-, and GAA-rich genes. (A) Schematic representation of the quantitative proteomics workflow used to assess differential translation in urm1∆ cells. (B) Volcano plot of protein abundance ratios vs. their bayes normalized t test calculated confidence. Results shown are of six biological replicates. Red dotted line: 5% FDR chosen as statistical significance. (C) Barplot representation of the variable importance learned by a random forest algorithm used to predict the ability of different codon abundances in classifying proteins in significantly up- and down-regulated sets. Dotted line: absolute value of the lowest predictor. (D) Proteins with the corresponding highest frequency (1% of the genome) of AAA, CAA, and GAA codons represented in the volcano plot from Fig. 2B. Red dotted line: 5% FDR. (E) Heatmap of log₂(WT/urm1∆) protein abundance ratios of the significantly up- and down-regulated proteins from Fig. 2B in cells overexpressing tK^UUU, tQ^UUG, and tE^UUC individually or in combination compared with cells without plasmid. Columns are clustered based on Euclidean distance. Figs. S1–S3.

Fig. 3.

The URM1-pathway specifically regulates translation of Cms1 and Ypl199c. (A and B) Volcano plots of protein abundance ratios with statistical significance measured by quantitative proteomics of the top 1.5% yeast genes with the highest (A) AAA or (B) CAA codon frequency. (C) Western blot of TAP-tagged Cms1 or Ypl199c from wild-type and urm1∆ cells using PAP antibodies or anti-Pgk1 as loading control. The quantification indicates the urm1∆/WT protein abundance ratio averaged from three independent experiments. (D) The mRNA levels of CMS1 and YPL199C and the ACT1 or URM1 controls were measured by quantitative PCR in wild-type and urm1∆ cells. Data show the mean ± SEM of three independent experiments. (E and F) The protein stability of TAP-tagged (E) Cms1 and (F) Ypl199c in wild-type and urm1∆ cells was determined by Western blot of extracts prepared at the indicated time points (min) after CHX addition. Protein amount over time were compared with amount at time 0. Data show the mean ± SEM of three independent experiments. See also Figs. S2 and S4.

Fig. 4.

URM1 is required for efficient translation of AAA-, GAA-, and CAA-enriched reporters. (A) Schematic representation of the dual-fluorescent codon-specific translation reporter. β-Estradiol–inducible quadruple-venus (4× YFP) or quadruple-CFP (4× CFP) proteins serve as codon-specific translation reporters and internal translation control, respectively. Codon-traps composed of a run of 10 identical codons, (XXX)₁₀, are inserted at the N-terminus of YFP. (B) Time course of (CAA)₁₀ translation reporter expression induced at time 0 in wild-type and urm1∆ cells. Data show the mean YFP/CFP ratio ± SEM from at least 100 cells plotted as percentage of maximum expression. (C and D) Expression of the translation reporter after 3-h induction with (C) (CAA)₁₀ codon-trap in different mutants or (D) different codon-traps in wild-type and urm1∆ cells. Data show the mean YFP/CFP ratio ± SEM from at least 1,000 cells plotted as percent of wild-type control. See also Fig. S5.

Fig. 5.

tRNA thiolation of U₃₄ promotes A-site binding and dipeptide formation in vitro. (A) Schematic illustration of the decoding and peptide bond formation processes. (B and C) A-site binding of [¹⁴C]Lys-tRNA^Lys (B) or for control [¹⁴C]Phe-tRNA^Phe (C) isolated from wild-type, urm1∆, or elp3∆ cells containing tK^UUU (mcm⁵s²U₃₄, mcm⁵U₃₄, or s²U₃₄ modifications) and tF^GAA, were measured after incubation of initiation complex with ternary complex. Data show the mean [¹⁴C] signal ± SEM of three independent experiments plotted as percentage of wild-type. (D) The rate constants of tRNA dissociation (k_off) and tRNA association (k_on) of peptidyl-tRNA^Lys prepared from wild-type, urm1∆, or elp3∆ cells. The equilibrium dissociation constant (K_d) ± SEM is shown below the graph. (E) The rate of dipeptide formation (k_pep) using tRNA^Lys isolated from wild-type and urm1∆ cells.