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: 11.47). 07/2012; 3:937. DOI: 10.1038/ncomms1938
Source: PubMed


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
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    • "Since lack of modifications is often overcome by increased dosage of one or more of the unmodified tRNAs (Esberg, et al., 2006; Fernandez-Vazquez, et al., 2013; Guy, et al., 2012; Han, et al., 2015; Phizicky and Alfonzo, 2010), the numerous links between tRNA modifications and neurological defects suggest that the available pool of functional tRNAs may somehow be limited during development and function of the central nervous system, presumably leading to defects in translation or its regulation (Begley, et al., 2007; Chan, et al., 2012). The specific mechanisms by which defects in tRNA biology impact neurological function remain to be determined. "
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    ABSTRACT: tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734 respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause non-syndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here we report that tRNA(Phe) from two genetically independent cell lines of NSXLID patients with loss of function FTSJ1 mutations nearly completely lacks Cm32 and Gm34 , and has reduced peroxywybutosine (o2yW37 ). Additionally, tRNA(Phe) from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34 , but has normal levels of Cm32 and o2yW37 . tRNA(Phe) from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34 , and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNA(Phe) as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNA(Phe) . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Human Mutation 08/2015; DOI:10.1002/humu.22897 · 5.14 Impact Factor
    • "With purified RNA in hand, the next step is to hydrolyze the RNA into oligonucleotide fragments or individual ribonucleotides, with the latter dephosphorylated to ribonucleoside form for LC–MS analysis. The oligonucleotides are used for localizing and quantifying modified ribonucleosides in specific tRNA species (Castleberry & Limbach, 2010; Chan et al., 2012; Hossain & Limbach, 2007), while the ribonucleosides can be identified and quantified by LC–MS as discussed later in this chapter. We focus here on the hydrolysis of RNA into ribonucleosides for analysis of stress-induced changes and patterns in translational response mechanisms. "
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    ABSTRACT: Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications. © 2015 Elsevier Inc. All rights reserved.
    Methods in enzymology 08/2015; 560:29-71. DOI:10.1016/bs.mie.2015.03.004 · 2.09 Impact Factor
    • "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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