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1
Huber, Sabrina, Andrea Leonardi, Peter Dedon, and Thomas Begley. "The Versatile Roles of the tRNA Epitranscriptome during Cellular Responses to Toxic Exposures and Environmental Stress." Toxics 7, no. 1 (March 25, 2019): 17. http://dx.doi.org/10.3390/toxics7010017.
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Living organisms respond to environmental changes and xenobiotic exposures by regulating gene expression. While heat shock, unfolded protein, and DNA damage stress responses are well-studied at the levels of the transcriptome and proteome, tRNA-mediated mechanisms are only recently emerging as important modulators of cellular stress responses. Regulation of the stress response by tRNA shows a high functional diversity, ranging from the control of tRNA maturation and translation initiation, to translational enhancement through modification-mediated codon-biased translation of mRNAs encoding stress response proteins, and translational repression by stress-induced tRNA fragments. tRNAs need to be heavily modified post-transcriptionally for full activity, and it is becoming increasingly clear that many aspects of tRNA metabolism and function are regulated through the dynamic introduction and removal of modifications. This review will discuss the many ways that nucleoside modifications confer high functional diversity to tRNAs, with a focus on tRNA modification-mediated regulation of the eukaryotic response to environmental stress and toxicant exposures. Additionally, the potential applications of tRNA modification biology in the development of early biomarkers of pathology will be highlighted.
2
Pereira, Marisa, Stephany Francisco, Ana Varanda, Mafalda Santos, Manuel Santos, and Ana Soares. "Impact of tRNA Modifications and tRNA-Modifying Enzymes on Proteostasis and Human Disease." International Journal of Molecular Sciences 19, no. 12 (November 24, 2018): 3738. http://dx.doi.org/10.3390/ijms19123738.
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Transfer RNAs (tRNAs) are key players of protein synthesis, as they decode the genetic information organized in mRNA codons, translating them into the code of 20 amino acids. To be fully active, tRNAs undergo extensive post-transcriptional modifications, catalyzed by different tRNA-modifying enzymes. Lack of these modifications increases the level of missense errors and affects codon decoding rate, contributing to protein aggregation with deleterious consequences to the cell. Recent works show that tRNA hypomodification and tRNA-modifying-enzyme deregulation occur in several diseases where proteostasis is affected, namely, neurodegenerative and metabolic diseases. In this review, we discuss the recent findings that correlate aberrant tRNA modification with proteostasis imbalances, in particular in neurological and metabolic disorders, and highlight the association between tRNAs, their modifying enzymes, translational decoding, and disease onset.
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Kazuhito, Tomizawa, and Fan-Yan Wei. "Posttranscriptional modifications in mitochondrial tRNA and its implication in mitochondrial translation and disease." Journal of Biochemistry 168, no. 5 (August 20, 2020): 435–44. http://dx.doi.org/10.1093/jb/mvaa098.
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Abstract A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. Mitochondrial DNA encodes 22 tRNAs that translate mitochondrial mRNAs to 13 polypeptides of respiratory complexes. Various chemical modifications have been identified in mitochondrial tRNAs via complex enzymatic processes. A growing body of evidence has demonstrated that these modifications are essential for translation by regulating tRNA stability, structure and mRNA binding, and can be dynamically regulated by the metabolic environment. Importantly, the hypomodification of mitochondrial tRNA due to pathogenic mutations in mitochondrial tRNA genes or nuclear genes encoding modifying enzymes can result in life-threatening mitochondrial diseases in humans. Thus, the mitochondrial tRNA modification is a fundamental mechanism underlying the tight regulation of mitochondrial translation and is essential for life. In this review, we focus on recent findings on the physiological roles of 5-taurinomethyl modification (herein referred as taurine modification) in mitochondrial tRNAs. We summarize the findings in human patients and animal models with a deficiency of taurine modifications and provide pathogenic links to mitochondrial diseases. We anticipate that this review will help understand the complexity of mitochondrial biology and disease.
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de Crécy-Lagard, Valérie, Robert L. Ross, Marshall Jaroch, Virginie Marchand, Christina Eisenhart, Damien Brégeon, Yuri Motorin, and Patrick A. Limbach. "Survey and Validation of tRNA Modifications and Their Corresponding Genes in Bacillus subtilis sp Subtilis Strain 168." Biomolecules 10, no. 7 (June 30, 2020): 977. http://dx.doi.org/10.3390/biom10070977.
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Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli. In general, B. subtilis tRNAs are less modified than those in E. coli, even if some modifications, such as m1A22 or ms2t6A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.
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Kimura, Satoshi, and Matthew K. Waldor. "The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA." Proceedings of the National Academy of Sciences 116, no. 4 (January 8, 2019): 1394–403. http://dx.doi.org/10.1073/pnas.1814130116.
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The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification ofVibrio choleraetRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiIstrain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence ofthiI. Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.
6
Galvanin, Adeline, Lea-Marie Vogt, Antonia Grober, Isabel Freund, Lilia Ayadi, Valerie Bourguignon-Igel, Larissa Bessler, et al. "Bacterial tRNA 2′-O-methylation is dynamically regulated under stress conditions and modulates innate immune response." Nucleic Acids Research 48, no. 22 (December 4, 2020): 12833–44. http://dx.doi.org/10.1093/nar/gkaa1123.
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Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.
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Strobel, M. C., and J. Abelson. "Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo." Molecular and Cellular Biology 6, no. 7 (July 1986): 2663–73. http://dx.doi.org/10.1128/mcb.6.7.2663-2673.1986.
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The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.
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Strobel, M. C., and J. Abelson. "Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo." Molecular and Cellular Biology 6, no. 7 (July 1986): 2663–73. http://dx.doi.org/10.1128/mcb.6.7.2663.
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The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.
9
Pereira, Marisa, Diana R. Ribeiro, Miguel M. Pinheiro, Margarida Ferreira, Stefanie Kellner, and Ana R. Soares. "m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs." International Journal of Molecular Sciences 22, no. 6 (March 14, 2021): 2941. http://dx.doi.org/10.3390/ijms22062941.
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Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.
10
Lei, Lei, and Zachary Frome Burton. "“Superwobbling” and tRNA-34 Wobble and tRNA-37 Anticodon Loop Modifications in Evolution and Devolution of the Genetic Code." Life 12, no. 2 (February 8, 2022): 252. http://dx.doi.org/10.3390/life12020252.
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The genetic code evolved around the reading of the tRNA anticodon on the primitive ribosome, and tRNA-34 wobble and tRNA-37 modifications coevolved with the code. We posit that EF-Tu, the closing mechanism of the 30S ribosomal subunit, methylation of wobble U34 at the 5-carbon and suppression of wobbling at the tRNA-36 position were partly redundant and overlapping functions that coevolved to establish the code. The genetic code devolved in evolution of mitochondria to reduce the size of the tRNAome (all of the tRNAs of an organism or organelle). “Superwobbling” or four-way wobbling describes a major mechanism for shrinking the mitochondrial tRNAome. In superwobbling, unmodified wobble tRNA-U34 can recognize all four codon wobble bases (A, G, C and U), allowing a single unmodified tRNA-U34 to read a 4-codon box. During code evolution, to suppress superwobbling in 2-codon sectors, U34 modification by methylation at the 5-carbon position appears essential. As expected, at the base of code evolution, tRNA-37 modifications mostly related to the identity of the adjacent tRNA-36 base. TRNA-37 modifications help maintain the translation frame during elongation.
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Khalique, Abdul, Sandy Mattijssen, and Richard J. Maraia. "A versatile tRNA modification-sensitive northern blot method with enhanced performance." RNA 28, no. 3 (December 20, 2021): 418–32. http://dx.doi.org/10.1261/rna.078929.121.
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The 22 mitochondrial and ∼45 cytosolic tRNAs in human cells contain several dozen different post-transcriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression, and deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our laboratory developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ∼10 yr ago, which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of a DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32, and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.
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Hopper, Anita K., and Hsiao-Yun Huang. "Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking." Molecular and Cellular Biology 35, no. 12 (April 6, 2015): 2052–58. http://dx.doi.org/10.1128/mcb.00131-15.
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tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair.
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Damon, Jadyn R., David Pincus, and Hidde L. Ploegh. "tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae." Molecular Biology of the Cell 26, no. 2 (January 15, 2015): 270–82. http://dx.doi.org/10.1091/mbc.e14-06-1145.
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Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas 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 tGluUUC, tGlnUUG, and tLysUUU 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.
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Tomikawa, Chie. "7-Methylguanosine Modifications in Transfer RNA (tRNA)." International Journal of Molecular Sciences 19, no. 12 (December 17, 2018): 4080. http://dx.doi.org/10.3390/ijms19124080.
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More than 90 different modified nucleosides have been identified in tRNA. Among the tRNA modifications, the 7-methylguanosine (m7G) modification is found widely in eubacteria, eukaryotes, and a few archaea. In most cases, the m7G modification occurs at position 46 in the variable region and is a product of tRNA (m7G46) methyltransferase. The m7G46 modification forms a tertiary base pair with C13-G22, and stabilizes the tRNA structure. A reaction mechanism for eubacterial tRNA m7G methyltransferase has been proposed based on the results of biochemical, bioinformatic, and structural studies. However, an experimentally determined mechanism of methyl-transfer remains to be ascertained. The physiological functions of m7G46 in tRNA have started to be determined over the past decade. For example, tRNA m7G46 or tRNA (m7G46) methyltransferase controls the amount of other tRNA modifications in thermophilic bacteria, contributes to the pathogenic infectivity, and is also associated with several diseases. In this review, information of tRNA m7G modifications and tRNA m7G methyltransferases is summarized and the differences in reaction mechanism between tRNA m7G methyltransferase and rRNA or mRNA m7G methylation enzyme are discussed.
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Meyer, Britta, Carina Immer, Steffen Kaiser, Sunny Sharma, Jun Yang, Peter Watzinger, Lena Weiß, et al. "Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs." Nucleic Acids Research 48, no. 3 (December 21, 2019): 1435–50. http://dx.doi.org/10.1093/nar/gkz1191.
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Abstract tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.
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Edwards, Ashley M., Maame A. Addo, and Patricia C. Dos Santos. "Extracurricular Functions of tRNA Modifications in Microorganisms." Genes 11, no. 8 (August 7, 2020): 907. http://dx.doi.org/10.3390/genes11080907.
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Transfer RNAs (tRNAs) are essential adaptors that mediate translation of the genetic code. These molecules undergo a variety of post-transcriptional modifications, which expand their chemical reactivity while influencing their structure, stability, and functionality. Chemical modifications to tRNA ensure translational competency and promote cellular viability. Hence, the placement and prevalence of tRNA modifications affects the efficiency of aminoacyl tRNA synthetase (aaRS) reactions, interactions with the ribosome, and transient pairing with messenger RNA (mRNA). The synthesis and abundance of tRNA modifications respond directly and indirectly to a range of environmental and nutritional factors involved in the maintenance of metabolic homeostasis. The dynamic landscape of the tRNA epitranscriptome suggests a role for tRNA modifications as markers of cellular status and regulators of translational capacity. This review discusses the non-canonical roles that tRNA modifications play in central metabolic processes and how their levels are modulated in response to a range of cellular demands.
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Yang, Wen-Qing, Qing-Ping Xiong, Jian-Yang Ge, Hao Li, Wen-Yu Zhu, Yan Nie, Xiuying Lin, et al. "THUMPD3–TRMT112 is a m2G methyltransferase working on a broad range of tRNA substrates." Nucleic Acids Research 49, no. 20 (October 20, 2021): 11900–11919. http://dx.doi.org/10.1093/nar/gkab927.
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Abstract Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m2G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m2G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3–TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3′-CCA of mature tRNAs. We also showed that m2G7 of tRNATrp was introduced by THUMPD3–TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3-knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m2G6/7 modification and pave a way for further studies of the role of m2G in sperm tRNA derived fragments.
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Tang, Jun, Pengfei Jia, Peiyong Xin, Jinfang Chu, Dong-Qiao Shi, and Wei-Cai Yang. "The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase." Journal of Experimental Botany 71, no. 10 (February 25, 2020): 3024–36. http://dx.doi.org/10.1093/jxb/eraa100.
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Abstract tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.
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Sun, Congliang, Patrick A. Limbach, and Balasubrahmanyam Addepalli. "Characterization of UVA-Induced Alterations to Transfer RNA Sequences." Biomolecules 10, no. 11 (November 8, 2020): 1527. http://dx.doi.org/10.3390/biom10111527.
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Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs.
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Hawer, Harmen, Alexander Hammermeister, Keerthiraju Ravichandran, Sebastian Glatt, Raffael Schaffrath, and Roland Klassen. "Roles of Elongator Dependent tRNA Modification Pathways in Neurodegeneration and Cancer." Genes 10, no. 1 (December 28, 2018): 19. http://dx.doi.org/10.3390/genes10010019.
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Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the emerging epitranscriptomic mRNA regulators. Several of the tRNA modifications are required to prevent human diseases and are particularly important for proper development and generation of neurons. In addition to the positive role of different tRNA modifications in prevention of neurodegeneration, certain cancer types upregulate tRNA modification genes to sustain cancer cell gene expression and metastasis. Multiple associations of defects in genes encoding subunits of the tRNA modifier complex Elongator with human disease highlight the importance of proper anticodon wobble uridine modifications (xm5U34) for health. Elongator functionality requires communication with accessory proteins and dynamic phosphorylation, providing regulatory control of its function. Here, we summarized recent insights into molecular functions of the complex and the role of Elongator dependent tRNA modification in human disease.
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Yoluç, Yasemin, Erik van de Logt, and Stefanie Kellner-Kaiser. "The Stress-Dependent Dynamics of Saccharomyces cerevisiae tRNA and rRNA Modification Profiles." Genes 12, no. 9 (August 28, 2021): 1344. http://dx.doi.org/10.3390/genes12091344.
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RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.
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Zhang, Jilei, Yong-guo Zhang, Yinglin Xia, and Jun Sun. "TRANSFER RNA MODIFICATION CONTRIBUTE TO PATHOGENESIS OF INTESTINAL INFLAMMATION BY ALTERING EPITHELIAL BARRIERS." Inflammatory Bowel Diseases 29, Supplement_1 (January 26, 2023): S55—S56. http://dx.doi.org/10.1093/ibd/izac247.107.
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Abstract BACKGROUNDS Transfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine (Q)-modification is a fundamental process for fidelity and efficiency of translation from RNA to proteins. In eukaryotes, tRNA-Q-modification relies on the intestinal microbial product queuine. Growing evidence indicates that tRNA modifications play important roles in human diseases, e.g., type 2 diabetes. Q depletion led to endoplasmic reticulum stress in mouse liver. Q-tRNA modifications are dynamic and highly variable depending on the developmental stages and species type and tumorigenesis. However, the health consequences of disturbed availability of queuine and altered Q-tRNA modification remain to be investigated. The effects and mechanisms of Q-tRNA in intestinal bowel diseases (IBD) are unknown. METHODS We explored the Q-tRNA-related tRNA synthetases and expression of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used several colitis mouse models, organoids, and cultured cells for loss- and gain-of-function studies to investigate the mechanisms of Q-tRNA modifications in intestinal barrier functions, proliferation, and inflammation. RESULTS In ulcerative colitis and Crohn’s disease patients, QTRT1 expression was significantly downregulated. Altered QTRT1-related metabolites were found in human IBD. The four Q-tRNA-related tRNA synthetases, asparaginyl-aspartyl-, histidyl-, and tyrosyl-tRNA synthetase, were decreased in IBD patients. This reduction was further confirmed in DSS-induced colitis and IL10-deficient mice. Reduced QTRT1 was significantly correlated with intestinal junctions, including downregulated β-catenin and upregulated Claudin-2. These alterations were confirmed in vitro by deleting QTRT1 from cells. Queuine treatment significantly enhanced cell junctional functions and reduced inflammation in epithelial cells. CONCLUSION tRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial barriers. Insights into Q-tRNA modification in regulating barrier functions would facilitate the development of targeted interventions for IBD through queuine and tRNA modifications.
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Fu, Dragony, Jennifer A. N. Brophy, Clement T. Y. Chan, Kyle A. Atmore, Ulrike Begley, Richard S. Paules, Peter C. Dedon, Thomas J. Begley, and Leona D. Samson. "Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival." Molecular and Cellular Biology 30, no. 10 (March 22, 2010): 2449–59. http://dx.doi.org/10.1128/mcb.01604-09.
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ABSTRACT tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm5U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm5U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.
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Klassen, Roland, Alexander Bruch, and Raffael Schaffrath. "Induction of protein aggregation and starvation response by tRNA modification defects." Current Genetics 66, no. 6 (August 29, 2020): 1053–57. http://dx.doi.org/10.1007/s00294-020-01103-w.
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Abstract Posttranscriptional modifications of anticodon loops contribute to the decoding efficiency of tRNAs by supporting codon recognition and loop stability. Consistently, strong synthetic growth defects are observed in yeast strains simultaneously lacking distinct anticodon loop modifications. These phenotypes are accompanied by translational inefficiency of certain mRNAs and disturbed protein homeostasis resulting in accumulation of protein aggregates. Different combinations of anticodon loop modification defects were shown to affect distinct tRNAs but provoke common transcriptional changes that are reminiscent of the cellular response to nutrient starvation. Multiple mechanisms may be involved in mediating inadequate starvation response upon loss of critical tRNA modifications. Recent evidence suggests protein aggregate induction to represent one such trigger.
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Kouvela, Adamantia, Apostolos Zaravinos, and Vassiliki Stamatopoulou. "Adaptor Molecules Epitranscriptome Reprograms Bacterial Pathogenicity." International Journal of Molecular Sciences 22, no. 16 (August 5, 2021): 8409. http://dx.doi.org/10.3390/ijms22168409.
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The strong decoration of tRNAs with post-transcriptional modifications provides an unprecedented adaptability of this class of non-coding RNAs leading to the regulation of bacterial growth and pathogenicity. Accumulating data indicate that tRNA post-transcriptional modifications possess a central role in both the formation of bacterial cell wall and the modulation of transcription and translation fidelity, but also in the expression of virulence factors. Evolutionary conserved modifications in tRNA nucleosides ensure the proper folding and stability redounding to a totally functional molecule. However, environmental factors including stress conditions can cause various alterations in tRNA modifications, disturbing the pathogen homeostasis. Post-transcriptional modifications adjacent to the anticodon stem-loop, for instance, have been tightly linked to bacterial infectivity. Currently, advances in high throughput methodologies have facilitated the identification and functional investigation of such tRNA modifications offering a broader pool of putative alternative molecular targets and therapeutic avenues against bacterial infections. Herein, we focus on tRNA epitranscriptome shaping regarding modifications with a key role in bacterial infectivity including opportunistic pathogens of the human microbiome.
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Hoffmann, Anne, Lieselotte Erber, Heike Betat, Peter F. Stadler, Mario Mörl, and Jörg Fallmann. "Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum." Non-Coding RNA 7, no. 2 (May 28, 2021): 32. http://dx.doi.org/10.3390/ncrna7020032.
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Dictyostelium discoideum is a social amoeba, which on starvation develops from a single-cell state to a multicellular fruiting body. This developmental process is accompanied by massive changes in gene expression, which also affect non-coding RNAs. Here, we investigate how tRNAs as key regulators of the translation process are affected by this transition. To this end, we used LOTTE-seq to sequence the tRNA pool of D. discoideum at different developmental time points and analyzed both tRNA composition and tRNA modification patterns. We developed a workflow for the specific detection of modifications from reverse transcriptase signatures in chemically untreated RNA-seq data at single-nucleotide resolution. It avoids the comparison of treated and untreated RNA-seq data using reverse transcription arrest patterns at nucleotides in the neighborhood of a putative modification site as internal control. We find that nucleotide modification sites in D. discoideum tRNAs largely conform to the modification patterns observed throughout the eukaroytes. However, there are also previously undescribed modification sites. We observe substantial dynamic changes of both expression levels and modification patterns of certain tRNA types during fruiting body development. Beyond the specific application to D. discoideum our results demonstrate that the developmental variability of tRNA expression and modification can be traced efficiently with LOTTE-seq.
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Keffer-Wilkes, Laura Carole, Govardhan Reddy Veerareddygari, and Ute Kothe. "RNA modification enzyme TruB is a tRNA chaperone." Proceedings of the National Academy of Sciences 113, no. 50 (November 14, 2016): 14306–11. http://dx.doi.org/10.1073/pnas.1607512113.
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Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB’s RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870–1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.
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Tuorto, Francesca, and Frank Lyko. "Genome recoding by tRNA modifications." Open Biology 6, no. 12 (December 2016): 160287. http://dx.doi.org/10.1098/rsob.160287.
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RNA modifications are emerging as an additional regulatory layer on top of the primary RNA sequence. These modifications are particularly enriched in tRNAs where they can regulate not only global protein translation, but also protein translation at the codon level. Modifications located in or in the vicinity of tRNA anticodons are highly conserved in eukaryotes and have been identified as potential regulators of mRNA decoding. Recent studies have provided novel insights into how these modifications orchestrate the speed and fidelity of translation to ensure proper protein homeostasis. This review highlights the prominent modifications in the tRNA anticodon loop: queuosine, inosine, 5-methoxycarbonylmethyl-2-thiouridine, wybutosine, threonyl–carbamoyl–adenosine and 5-methylcytosine. We discuss the functional relevance of these modifications in protein translation and their emerging role in eukaryotic genome recoding during cellular adaptation and disease.
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Biedenbänder, Thomas, Vanessa de Jesus, Martina Schmidt-Dengler, Mark Helm, Björn Corzilius, and Boris Fürtig. "RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics." Nucleic Acids Research 50, no. 4 (February 7, 2022): 2334–49. http://dx.doi.org/10.1093/nar/gkac040.
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Abstract A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.
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Gehrig, Stefanie, Mariel-Esther Eberle, Flavia Botschen, Katharina Rimbach, Florian Eberle, Tatjana Eigenbrod, Steffen Kaiser, et al. "Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity." Journal of Experimental Medicine 209, no. 2 (February 6, 2012): 225–33. http://dx.doi.org/10.1084/jem.20111044.
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Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNATyr from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5′-unphosphorylated in vitro transcript of tRNATyr induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNATyr variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2′-O-methylated Gm18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNAPhe also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks Gm18 2′-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2′-oxygen of Gm18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.
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Piskounova, Elena. "Abstract PR001: Codon-bias and tRNA modifications as drivers of melanoma metastasis." Cancer Research 83, no. 2_Supplement_2 (January 15, 2023): PR001. http://dx.doi.org/10.1158/1538-7445.metastasis22-pr001.
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Abstract Only tumor cells that are able to enhance their stress response pathways, are able to successfully metastasize and colonize distant organs. In yeast, reprogramming of translation through modification of the tRNA anticodon loop in response to stress has been shown to be a central adaptive mechanism of survival. Distinct tRNA modifications increase translational efficiency of certain codons. These modifications are upregulated and are essential for survival under a range of stressors, including oxidative stress. The adaptive proteome is therefore encoded by specific transcripts with a distinct codon bias and regulated by these tRNA modifications. We have identified several stress-induced tRNA anticodon loop modifications in metastasizing melanoma cells that drive codon-biased translation of the adaptive pro-metastatic proteome. One of these modifications specifically regulates stress-induced translation of selenocysteine-containing proteins. Selenoproteins are central to stress resistance including oxidative and proteotoxic stress. We have identified and characterized the methyltransferase that regulates this modification and enables translation of the metastatic selenoproteome. More broadly, by combining biochemical approaches with in vivo models of melanoma metastasis, we have now identified several other enzymes responsible for multiple tRNA anticodon modifications as novel and targetable drivers of metastatic disease. Our work aims to establish how tRNA anticodon modifications and a distinct codon bias reprogram the metastatic proteome as a therapeutically targetable stress response mechanism. Citation Format: Elena Piskounova. Codon-bias and tRNA modifications as drivers of melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR001.
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Ramírez, Vicente, Beatriz González, Ana López, Maria Jose Castelló, Maria José Gil, Bo Zheng, Peng Chen, and Pablo Vera. "A 2′-O-Methyltransferase Responsible for Transfer RNA Anticodon Modification Is Pivotal for Resistance to Pseudomonas syringae DC3000 in Arabidopsis." Molecular Plant-Microbe Interactions® 31, no. 12 (December 2018): 1323–36. http://dx.doi.org/10.1094/mpmi-06-18-0148-r.
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Transfer RNA (tRNA) is the most highly modified class of RNA species in all living organisms. Recent discoveries have revealed unprecedented complexity in the tRNA chemical structures, modification patterns, regulation, and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge of the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2′-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance susceptibility during infection with the virulent bacterial pathogen Pseudomonas syringae DC3000. Lack of such tRNA modification, as observed in scs9 mutants, specifically dampens plant resistance against DC3000 without compromising the activation of the salicylic acid signaling pathway or the resistance to other biotrophic pathogens. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective disease resistance in Arabidopsis toward DC3000 and, therefore, expands the repertoire of molecular components essential for an efficient disease resistance response.
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Fages-Lartaud, Maxime, and Martin Frank Hohmann-Marriott. "Overview of tRNA Modifications in Chloroplasts." Microorganisms 10, no. 2 (January 20, 2022): 226. http://dx.doi.org/10.3390/microorganisms10020226.
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The chloroplast is a promising platform for biotechnological innovation due to its compact translation machinery. Nucleotide modifications within a minimal set of tRNAs modulate codon–anticodon interactions that are crucial for translation efficiency. However, a comprehensive assessment of these modifications does not presently exist in chloroplasts. Here, we synthesize all available information concerning tRNA modifications in the chloroplast and assign translation efficiency for each modified anticodon–codon pair. In addition, we perform a bioinformatics analysis that links enzymes to tRNA modifications and aminoacylation in the chloroplast of Chlamydomonas reinhardtii. This work provides the first comprehensive analysis of codon and anticodon interactions of chloroplasts and its implication for translation efficiency.
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Noon, Kathleen R., Rebecca Guymon, Pamela F. Crain, James A. McCloskey, Michael Thomm, Julianne Lim, and Ricardo Cavicchioli. "Influence of Temperature on tRNA Modification in Archaea: Methanococcoides burtonii (Optimum Growth Temperature [Topt], 23°C) and Stetteria hydrogenophila (Topt, 95°C)." Journal of Bacteriology 185, no. 18 (September 15, 2003): 5483–90. http://dx.doi.org/10.1128/jb.185.18.5483-5490.2003.
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ABSTRACT We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23°C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93°C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2′ of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.
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Kulkarni, Sneha, Mary Anne T. Rubio, Eva Hegedűsová, Robert L. Ross, Patrick A. Limbach, Juan D. Alfonzo, and Zdeněk Paris. "Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis." Nucleic Acids Research 49, no. 14 (July 9, 2021): 8247–60. http://dx.doi.org/10.1093/nar/gkab567.
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Abstract Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.
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Lampi, Mirka, Pavlina Gregorova, M. Suleman Qasim, Niklas C. V. Ahlblad, and L. Peter Sarin. "Bacteriophage Infection of the Marine Bacterium Shewanella glacialimarina Induces Dynamic Changes in tRNA Modifications." Microorganisms 11, no. 2 (January 31, 2023): 355. http://dx.doi.org/10.3390/microorganisms11020355.
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Viruses are obligate intracellular parasites that, throughout evolution, have adapted numerous strategies to control the translation machinery, including the modulation of post-transcriptional modifications (PTMs) on transfer RNA (tRNA). PTMs are critical translation regulators used to further host immune responses as well as the expression of viral proteins. Yet, we lack critical insight into the temporal dynamics of infection-induced changes to the tRNA modification landscape (i.e., ‘modificome’). In this study, we provide the first comprehensive quantitative characterization of the tRNA modificome in the marine bacterium Shewanella glacialimarina during Shewanella phage 1/4 infection. Specifically, we show that PTMs can be grouped into distinct categories based on modification level changes at various infection stages. Furthermore, we observe a preference for the UAC codon in viral transcripts expressed at the late stage of infection, which coincides with an increase in queuosine modification. Queuosine appears exclusively on tRNAs with GUN anticodons, suggesting a correlation between phage codon usage and PTM modification. Importantly, this work provides the basis for further studies into RNA-based regulatory mechanisms employed by bacteriophages to control the prokaryotic translation machinery.
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Funk, Holly M., Ruoxia Zhao, Maggie Thomas, Sarah M. Spigelmyer, Nichlas J. Sebree, Regan O. Bales, Jamison B. Burchett, Justen B. Mamaril, Patrick A. Limbach, and Michael P. Guy. "Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants." PLOS ONE 15, no. 11 (November 30, 2020): e0242737. http://dx.doi.org/10.1371/journal.pone.0242737.
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Posttranscriptional modification of tRNA is critical for efficient protein translation and proper cell growth, and defects in tRNA modifications are often associated with human disease. Although most of the enzymes required for eukaryotic tRNA modifications are known, many of these enzymes have not been identified and characterized in several model multicellular eukaryotes. Here, we present two related approaches to identify the genes required for tRNA modifications in multicellular organisms using primer extension assays with fluorescent oligonucleotides. To demonstrate the utility of these approaches we first use expression of exogenous genes in yeast to experimentally identify two TRM1 orthologs capable of forming N2,N2-dimethylguanosine (m2,2G) on residue 26 of cytosolic tRNA in the model plant Arabidopsis thaliana. We also show that a predicted catalytic aspartate residue is required for function in each of the proteins. We next use RNA interference in cultured Drosophila melanogaster cells to identify the gene required for m2,2G26 formation on cytosolic tRNA. Additionally, using these approaches we experimentally identify D. melanogaster gene CG10050 as the corresponding ortholog of human DTWD2, which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp3U) on residue 20a of cytosolic tRNA. We further show that A. thaliana gene AT2G41750 can form acp3U20b on an A. thaliana tRNA expressed in yeast cells, and that the aspartate and tryptophan residues in the DXTW motif of this protein are required for modification activity. These results demonstrate that these approaches can be used to study tRNA modification enzymes.
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Bruch, Alexander, Roland Klassen, and Raffael Schaffrath. "Unfolded Protein Response Suppression in Yeast by Loss of tRNA Modifications." Genes 9, no. 11 (October 23, 2018): 516. http://dx.doi.org/10.3390/genes9110516.
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Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyze whether this includes the activation of the unfolded protein response (UPR), which is commonly triggered by protein aggregation within the endoplasmic reticulum (ER). We demonstrate that two different aggregation prone tRNA modification mutants (elp6 ncs2; elp3 deg1) lacking combinations of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U: elp3; elp6; ncs2) and pseudouridine (Ψ: deg1) reduce, rather than increase, splicing of HAC1 mRNA, an event normally occurring as a precondition of UPR induction. In addition, tunicamycin (TM) induced HAC1 splicing is strongly impaired in the elp3 deg1 mutant. Strikingly, this mutant displays UPR independent resistance against TM, a phenotype we found to be rescued by overexpression of tRNAGln(UUG), the tRNA species usually carrying the mcm5s2U34 and Ψ38 modifications. Our data indicate that proper tRNA anticodon loop modifications promote rather than impair UPR activation and reveal that protein synthesis and homeostasis defects in their absence do not routinely result in UPR induction but may relieve endogenous ER stress.
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Urbonavičius, Jaunius, Jérôme M. B. Durand, and Glenn R. Björk. "Three Modifications in the D and T Arms of tRNA Influence Translation in Escherichia coli and Expression of Virulence Genes in Shigella flexneri." Journal of Bacteriology 184, no. 19 (October 1, 2002): 5348–57. http://dx.doi.org/10.1128/jb.184.19.5348-5357.2002.
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ABSTRACT The modified nucleosides 2′-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m5U54), and pseudouridine, present at position 55 (Ψ55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli. The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m5U54), and truB (Ψ55). The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-dl-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Ψ55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Ψ55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes.
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Delaunay, Sylvain, Francesca Rapino, Lars Tharun, Zhaoli Zhou, Lukas Heukamp, Martin Termathe, Kateryna Shostak, et al. "Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer." Journal of Experimental Medicine 213, no. 11 (October 10, 2016): 2503–23. http://dx.doi.org/10.1084/jem.20160397.
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Quantitative and qualitative changes in mRNA translation occur in tumor cells and support cancer progression and metastasis. Posttranscriptional modifications of transfer RNAs (tRNAs) at the wobble uridine 34 (U34) base are highly conserved and contribute to translation fidelity. Here, we show that ELP3 and CTU1/2, partner enzymes in U34 mcm5s2-tRNA modification, are up-regulated in human breast cancers and sustain metastasis. Elp3 genetic ablation strongly impaired invasion and metastasis formation in the PyMT model of invasive breast cancer. Mechanistically, ELP3 and CTU1/2 support cellular invasion through the translation of the oncoprotein DEK. As a result, DEK promotes the IRES-dependent translation of the proinvasive transcription factor LEF1. Consistently, a DEK mutant, whose codon composition is independent of U34 mcm5s2-tRNA modification, escapes the ELP3- and CTU1-dependent regulation and restores the IRES-dependent LEF1 expression. Our results demonstrate that the key role of U34 tRNA modification is to support specific translation during breast cancer progression and highlight a functional link between tRNA modification– and IRES-dependent translation during tumor cell invasion and metastasis.
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van Dooren, Sander H. J., Reinout Raijmakers, Helma Pluk, Angelique M. C. Lokate, Tom S. Koemans, Richelle E. C. Spanjers, Albert J. R. Heck, Wilbert C. Boelens, Walther J. van Venrooij, and Ger J. M. Pruijn. "Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity." Biochemistry and Cell Biology 89, no. 6 (December 2011): 545–53. http://dx.doi.org/10.1139/o11-055.
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The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.
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Szczupak, Patrycja, Malgorzata Sierant, Ewelina Wielgus, Ewa Radzikowska-Cieciura, Katarzyna Kulik, Agnieszka Krakowiak, Paulina Kuwerska, Grazyna Leszczynska, and Barbara Nawrot. "Escherichia coli tRNA 2-Selenouridine Synthase (SelU): Elucidation of Substrate Specificity to Understand the Role of S-Geranyl-tRNA in the Conversion of 2-Thio- into 2-Selenouridines in Bacterial tRNA." Cells 11, no. 9 (May 2, 2022): 1522. http://dx.doi.org/10.3390/cells11091522.
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The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions.
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Torres, Adrian Gabriel, Oscar Reina, Camille Stephan-Otto Attolini, and Lluís Ribas de Pouplana. "Differential expression of human tRNA genes drives the abundance of tRNA-derived fragments." Proceedings of the National Academy of Sciences 116, no. 17 (April 8, 2019): 8451–56. http://dx.doi.org/10.1073/pnas.1821120116.
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The human genome encodes hundreds of transfer RNA (tRNA) genes but their individual contribution to the tRNA pool is not fully understood. Deep sequencing of tRNA transcripts (tRNA-Seq) can estimate tRNA abundance at single gene resolution, but tRNA structures and posttranscriptional modifications impair these analyses. Here we present a bioinformatics strategy to investigate differential tRNA gene expression and use it to compare tRNA-Seq datasets from cultured human cells and human brain. We find that sequencing caveats affect quantitation of only a subset of human tRNA genes. Unexpectedly, we detect several cases where the differences in tRNA expression among samples do not involve variations at the level of isoacceptor tRNA sets (tRNAs charged with the same amino acid but using different anticodons), but rather among tRNA genes within the same isodecoder set (tRNAs having the same anticodon sequence). Because isodecoder tRNAs are functionally equal in terms of genetic translation, their differential expression may be related to noncanonical tRNA functions. We show that several instances of differential tRNA gene expression result in changes in the abundance of tRNA-derived fragments (tRFs) but not of mature tRNAs. Examples of differentially expressed tRFs include PIWI-associated RNAs, tRFs present in tissue samples but not in cells cultured in vitro, and somatic tissue-specific tRFs. Our data support that differential expression of tRNA genes regulate noncanonical tRNA functions performed by tRFs.
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Oberbauer, Vera, and Matthias Schaefer. "tRNA-Derived Small RNAs: Biogenesis, Modification, Function and Potential Impact on Human Disease Development." Genes 9, no. 12 (December 5, 2018): 607. http://dx.doi.org/10.3390/genes9120607.
Full textAbstract:
Transfer RNAs (tRNAs) are abundant small non-coding RNAs that are crucially important for decoding genetic information. Besides fulfilling canonical roles as adaptor molecules during protein synthesis, tRNAs are also the source of a heterogeneous class of small RNAs, tRNA-derived small RNAs (tsRNAs). Occurrence and the relatively high abundance of tsRNAs has been noted in many high-throughput sequencing data sets, leading to largely correlative assumptions about their potential as biologically active entities. tRNAs are also the most modified RNAs in any cell type. Mutations in tRNA biogenesis factors including tRNA modification enzymes correlate with a variety of human disease syndromes. However, whether it is the lack of tRNAs or the activity of functionally relevant tsRNAs that are causative for human disease development remains to be elucidated. Here, we review the current knowledge in regard to tsRNAs biogenesis, including the impact of RNA modifications on tRNA stability and discuss the existing experimental evidence in support for the seemingly large functional spectrum being proposed for tsRNAs. We also argue that improved methodology allowing exact quantification and specific manipulation of tsRNAs will be necessary before developing these small RNAs into diagnostic biomarkers and when aiming to harness them for therapeutic purposes.
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Tasak, Monika, and Eric M. Phizicky. "Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay path way in evolutionarily distant yeast species." PLOS Genetics 18, no. 7 (July 28, 2022): e1010215. http://dx.doi.org/10.1371/journal.pgen.1010215.
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All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5’-3’ exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3’-5’ exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.
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Schultz, Sarah Kai-Leigh, and Ute Kothe. "tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA." RNA 26, no. 9 (May 8, 2020): 1131–42. http://dx.doi.org/10.1261/rna.075473.120.
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Songe-Møller, Lene, Erwin van den Born, Vibeke Leihne, Cathrine B. Vågbø, Terese Kristoffersen, Hans E. Krokan, Finn Kirpekar, Pål Ø. Falnes, and Arne Klungland. "Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding." Molecular and Cellular Biology 30, no. 7 (February 1, 2010): 1814–27. http://dx.doi.org/10.1128/mcb.01602-09.
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ABSTRACT Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8 −/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8 −/− mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8 −/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.
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Kelley, Melissa, Mellie June Paulines, George Yoshida, Ryan Myers, Manasses Jora, Joel P. Levoy, Balasubrahmanyam Addepalli, Joshua B. Benoit, and Patrick A. Limbach. "Ionizing radiation and chemical oxidant exposure impacts on Cryptococcus neoformans transfer RNAs." PLOS ONE 17, no. 3 (March 29, 2022): e0266239. http://dx.doi.org/10.1371/journal.pone.0266239.
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Cryptococcus neoformans is a fungus that is able to survive abnormally high levels of ionizing radiation (IR). The radiolysis of water by IR generates reactive oxygen species (ROS) such as H2O2 and OH-. C. neoformans withstands the damage caused by IR and ROS through antioxidant production and enzyme-catalyzed breakdown of ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications to maintain their structure, stability, and/or function under such environmental conditions. Here, we investigated the effects of IR and H2O2 exposure on tRNAs in C. neoformans. We experimentally identified the modified nucleosides present in C. neoformans tRNAs and quantified changes in those modifications upon exposure to oxidative conditions. To better understand these modified nucleoside results, we also evaluated tRNA pool composition in response to the oxidative conditions. We found that regardless of environmental conditions, tRNA modifications and transcripts were minimally affected. A rationale for the stability of the tRNA pool and its concomitant profile of modified nucleosides is proposed based on the lack of codon bias throughout the C. neoformans genome and in particular for oxidative response transcripts. Our findings suggest that C. neoformans can rapidly adapt to oxidative environments as mRNA translation/protein synthesis are minimally impacted by codon bias.
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Khonsari, Bahar, Roland Klassen, and Raffael Schaffrath. "Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation." International Journal of Molecular Sciences 22, no. 16 (August 15, 2021): 8753. http://dx.doi.org/10.3390/ijms22168753.
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Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.
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Xue, Hong, Yilong Xue, Sylvie Doublié, and Charles W. Carter, Jr. "Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase." Biochemistry and Cell Biology 75, no. 6 (December 1, 1997): 709–15. http://dx.doi.org/10.1139/o97-054.
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A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure. Key words: aminoacyl-tRNA synthetase, Bacillus subtilis, chemical modification, conformational change, transfer RNA, tryptophanyl-tRNA synthetase.