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. 2012 Jul 3:3:937.
doi: 10.1038/ncomms1938.

Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins

Clement T Y Chan  1 , Yan Ling Joy Pang, Wenjun Deng, I Ramesh Babu, Madhu Dyavaiah, Thomas J Begley, Peter C Dedon
Affiliations

Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins

Clement T Y Chan et al. Nat Commun. .

Abstract

Selective translation of survival proteins is an important facet of the cellular stress response. We recently demonstrated that this translational control involves a stress-specific reprogramming of modified ribonucleosides in tRNA. Here we report the discovery of a step-wise translational control mechanism responsible for survival following oxidative stress. In yeast exposed to hydrogen peroxide, there is a Trm4 methyltransferase-dependent increase in the proportion of tRNA(Leu(CAA)) containing m(5)C at the wobble position, which causes selective translation of mRNA from genes enriched in the TTG codon. Of these genes, oxidative stress increases protein expression from the TTG-enriched ribosomal protein gene RPL22A, but not its unenriched paralogue. Loss of either TRM4 or RPL22A confers hypersensitivity to oxidative stress. Proteomic analysis reveals that oxidative stress causes a significant translational bias towards proteins coded by TTG-enriched genes. These results point to stress-induced reprogramming of tRNA modifications and consequential reprogramming of ribosomes in translational control of cell survival.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
H2O2 exposure increases the level of m5C at the wobble position of tRNALeu(CAA). (A) tRNALeu(CAA) was digested with ribonucleases to generate oligoribonucleotides containing m5C or C at position 34 (CAAG) or position 48 (UUCAA), and the oligoribonucleotides were quantified by mass spectrometry. (B) The graph shows the ratio of m5C/C in tRNALeu(CAA) from H2O2-treated cells relative to untreated cells. The data represent mean ± SD for three experiments. The data for position 34 are significantly different from those for position 48 by Student’s t-test with p < 0.05.
Figure 2
Figure 2
H2O2 and Trm4 methyltransferase control gene expression at the level of TTG codon usage. (A) Scheme illustrating the dual luciferase reporter system for assessing the effect of TTG codon usage on protein expression in wild-type and trm4Δ mutant yeast cells transformed with either control or 4X-TTG reporter plasmids. (B) Control and 4X-TTG reporter activity was quantified in H2O2-exposed (gray bars) and unexposed (white bars) wild-type or trm4Δ cells. The ratio of treated to untreated is indicated above each condition. Data represent mean ± deviation about the mean for three biological replicates.
Figure 3
Figure 3
Loss of Trm4 methyltransferase decreases the proportion of ribosomes containing TTG codon-enriched ribosomal protein paralogs. Ribosomal proteins in wild-type and trm4Δ mutant S. cerevisiae were quantified by LC-MS/MS (schematic inset) and the relative quantities of ribosomal protein paralogs presented as the ratio of the signal intensity for the paralog with high TTG-usage to that of the low-usage paralog. Data represent mean ± SD for three biological replicates. p values denote statistically significant differences by Student’s t-test.
Figure 4
Figure 4
H2O2 exposure increases the proportion of ribosomes containing ribosomal protein paralog Rpl22a in wild-type S. cerevisiae but not trm4Δ mutants. Cells were exposed to 2 mM H2O2 for 1 hr and the quantities of ribosomal proteins were determined by LC-MS/MS analysis. Data are expressed as the ratio of the TTG-enriched paralog Rpl22a to unenriched Rpl22b. Data represent mean ± SD of three biological replicates. Asterisks denote statistically significant differences between H2O2-treated and untreated cells as judged by Student’s t-test with p < 0.05.
Figure 5
Figure 5
SILAC-based proteomic analysis reveals that H2O2 enhances the translation of TTG-enriched proteins. Protein extracts from control and H2O2-treated wild-type and trm4 mutant yeast were mixed 1:1 with proteins from U-[13C,15N]-lysine-labeled lys1Δ yeast cells as an internal standard. Protein mixtures were then subjected to trypsin digestion and proteomic analysis by LC-QTOF analysis. The quantities of the 261 most abundant proteins appearing in each of four biological replicates were analyzed by Student’s t-test (p <0.05) for increased (up-regulation), decreased (down-regulation) or unchanged levels in H2O2-treated versus control cells, or wild-type versus trm4Δ mutant. Within these three groups of proteins, the frequency of using TTG to code for leucine was calculated. The resulting frequency data are presented as a box-and-whiskers plot with the bar representing the median value, the box encompassing the range of data between the first and third quartile, and the error bars embracing data within 1.5-times interquartile range. Differences between up-regulated, down-regulated and unchanged categories were subjected to Student’s t-test with the indicated p values.
Figure 6
Figure 6
Ribosomal protein paralog Rpl22a confers resistance to H2O2 exposure in S. cerevisiae. Wild-type S. cerevisiae and strains lacking RPL16A, RPL16B, RPL22A, or RPL22B were exposed to 5 mM H2O2 and survival was assayed as described in Methods. Data represent mean ± SD of three biological replicates. The asterisk denotes a statistically significant difference compared to all other values in the figure, as judged by Student’s t-test with p < 0.05.
Figure 7
Figure 7
Proposed mechanism by which increase in m5C level regulates translation of ribosomal protein paralogs and confers resistance to H2O2. Exposure to H2O2 leads to an elevation in the level of m5C at the wobble position of the leucine tRNA for translating the codon UUG on mRNA (A), which enhances the translation of the UUG-enriched RPL22A mRNA relative to its paralog RPL22B (B) and leads to changes in ribosome composition (C). This reprogramming of tRNA and ribosomes ultimately causes selective translation of proteins from genes enriched with the codon TTG.
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