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

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Nature Communications (Impact Factor: 10.74). 07/2012; 3:937. DOI: 10.1038/ncomms1938
Source: PubMed

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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Available from: Madhu Dyavaiah, Mar 04, 2014
    • "It follows that m 5 C is neither ubiquitous nor a constitutive RNA modification (with the exception of its presence in some abundant noncoding RNAs), which should be taken into consideration when planning to use RNA-BisSeq for (cytosine-5) RNA methylation discovery. In addition, m 5 C might mark RNA only under specific environmental or stress conditions (Becker et al., 2012; B€ ugl et al., 2000; Chan et al., 2010, 2012), which should guide the experimental design prior to employing RNA-BisSeq. Although RNA-BisSeq can reveal the exact position of m 5 C in a given RNA molecule, the use of this method is presently curtailed by the need to efficiently denature purified RNA preparations (a prerequisite for quantitative deamination) as well as by the harsh deamination conditions. "
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    ABSTRACT: Cells have developed molecular machineries, which can chemically modify DNA and RNA nucleosides. One particular and chemically simple modification, (cytosine-5) methylation (m5C), has been detected both in RNA and DNA suggesting universal use of m5C for the function of these nucleotide polymers. m5C can be reproducibly mapped to abundant noncoding RNAs (transfer RNA, tRNA and ribosomal RNA, rRNA), and recently, also nonabundant RNAs (including mRNAs) have been reported to carry this modification. Quantification of m5C content in total RNA preparations indicates that a limited number of RNAs carry this modification and suggests specific functions for (cytosine-5) RNA methylation. What exactly is the biological function of m5C in RNA? Before attempting to address this question, m5C needs to be mapped specifically and reproducibly, preferably on a transcriptome-wide scale. To facilitate the detection of m5C in its sequence context, RNA bisulfite sequencing (RNA-BisSeq) has been developed. This method relies on the efficient chemical deamination of nonmethylated cytosine, which can be read out as single nucleotide polymorphism (nonmethylated cytosine as thymine vs. methylated cytosine as cytosine), when differentially comparing cDNA libraries to reference sequences after DNA sequencing. Here, the basic protocol of RNA-BisSeq, its current applications and limitations are described.
    RNA modifications, Edited by Chuan He, 05/2015: chapter Chapter Fourteen - RNA 5-Methylcytosine Analysis by Bisulfite Sequencing: pages 297-329; Elsevier.
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    • "Methylation of tRNA has been documented to have regulatory roles in translation including aminoacylation [9], codon preferences [10] [11], preservation of reading frame [12] [13] and tRNA charging [14]. Defective tRNA modifications can lead to cell damage and diseases, for example in " Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes " (MELAS) an erroneous modification of the tRNA Leu leads to a disturbance of codoneanticodon interaction [15]. "
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    ABSTRACT: Methylation of tRNA is an important post-transcriptional modification and aberrations in tRNA modification has been implicated in cancer. The DNMT2 protein methylates C38 of tRNA-Asp and it has a role in cellular physiology and stress response and its expression levels are altered in cancer tissues. Here we studied whether DNMT2 somatic mutations found in cancer tissues affect the activity of the enzyme. We have generated 13 DNMT2 variants and purified the corresponding proteins. All proteins were properly folded as determined by circular dichroism spectroscopy. We tested their RNA methylation activity using in vitro generated tRNA-Asp. One of the mutations (E63K) caused a twofold increase in activity, while two of them led to a stronger (over fourfold decrease) in activity (G155S and L257V). Two additional mutant proteins were almost inactive (R371H and G155V). The strong effect of some of the somatic cancer mutations on DNMT2 activity suggests that these mutations have a functional role in tumorigenesis. Copyright © 2015. Published by Elsevier B.V.
    Biochimie 03/2015; 112. DOI:10.1016/j.biochi.2015.02.022 · 3.12 Impact Factor
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    • "URM1 pathway mutants display a variety of phenotypes, including increased sensitivity to oxidative stressors and defects in nutrient sensing and invasive growth, many of which are linked to defects in tRNA modification (Goehring et al., 2003a,b; Rubio-Texeira, 2007; Leidel et al., 2009). Along with recent studies demonstrating that the levels of certain tRNA modifications change in response to different growth conditions (Kamenski et al., 2007; Chan et al., 2010; Preston et al., 2012), the phenotypes of mutant cells suggest that the dynamic regulation of tRNA modification pathways plays an underappreciated role in the response of cells to a variety of stresses. Not much is known about the specific conditions that lead to changes in tRNA modification levels, the mechanisms that might regulate tRNA modifications, or the properties of differentially modified tRNAs. "
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    ABSTRACT: Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. While tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu(UUC), tGln(UUG) and tLys(UUU) in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable, and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need.
    Molecular Biology of the Cell 11/2014; 26(2). DOI:10.1091/mbc.E14-06-1145 · 4.55 Impact Factor
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