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Pathways to disease from natural variations in human cytoplasmic tRNAs

  • Jeremy T. Lant
    Footnotes
    Affiliations
    From the Departments of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
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  • Matthew D. Berg
    Footnotes
    Affiliations
    From the Departments of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
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  • Ilka U. Heinemann
    Affiliations
    From the Departments of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
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  • Christopher J. Brandl
    Affiliations
    From the Departments of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
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  • Patrick O’Donoghue
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Departments of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada

    Chemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
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  • Author Footnotes
    1 Recipient of a Postgraduate Studies Doctoral Scholarship and supported by the Natural Sciences and Engineering Research Council of Canada.
    2 Recipient of a(Canada Graduate Scholarship and supported by the Natural Sciences and Engineering Research Council of Canada and by generous donations from Graham Wright and James Robertson.
    4 The abbreviations used are: AARSaminoacyl-tRNA synthetaseSNPsingle nucleotide polymorphismTAMtranscription-associated mutagenesisCFTRcystic fibrosis transmembrane conductance regulatormcm5U5-methoxycarbonylmethyluridinemcm5Um5-methoxycarbonylmethyl-2′-O-methyluridineHIFhypoxia-inducible factorGtRNAdbGenomic tRNA databasepol IIIpolymerase IIIHDHuntington's disease.
Open AccessPublished:January 14, 2019DOI:https://doi.org/10.1074/jbc.REV118.002982
      Perfectly accurate translation of mRNA into protein is not a prerequisite for life. Resulting from errors in protein synthesis, mistranslation occurs in all cells, including human cells. The human genome encodes >600 tRNA genes, providing both the raw material for genetic variation and a buffer to ensure that resulting translation errors occur at tolerable levels. On the basis of data from the 1000 Genomes Project, we highlight the unanticipated prevalence of mistranslating tRNA variants in the human population and review studies on synthetic and natural tRNA mutations that cause mistranslation or de-regulate protein synthesis. Although mitochondrial tRNA variants are well known to drive human diseases, including developmental disorders, few studies have revealed a role for human cytoplasmic tRNA mutants in disease. In the context of the unexpectedly large number of tRNA variants in the human population, the emerging literature suggests that human diseases may be affected by natural tRNA variants that cause mistranslation or de-regulate tRNA expression and nucleotide modification. This review highlights examples relevant to genetic disorders, cancer, and neurodegeneration in which cytoplasmic tRNA variants directly cause or exacerbate disease and disease-linked phenotypes in cells, animal models, and humans. In the near future, tRNAs may be recognized as useful genetic markers to predict the onset or severity of human disease.

      Introduction

      Protein synthesis is an evolutionarily conserved process that is required by all life. In the interpretation of the genetic code, transfer RNAs (tRNAs) play a central role as they physically link amino acids to codons. Crick’s adaptor hypothesis predicted the existence of tRNAs in 1955: “each amino acid would combine chemically, at a special enzyme, with a small molecule which, having a specific hydrogen-bonding surface, would combine specifically with the nucleic acid template.” (
      • Crick F.H.C.
      On degenerate templates and the adaptor hypothesis.
      ). Just 3 years later, the first tRNAs were discovered as soluble RNAs involved in protein synthesis (
      • Hoagland M.B.
      • Stephenson M.L.
      • Scott J.F.
      • Hecht L.I.
      • Zamecnik P.C.
      A soluble ribonucleic acid intermediate in protein synthesis.
      ). Working with yeast, Holley et al. (
      • Holley R.W.
      • Everett G.A.
      • Madison J.T.
      • Zamir A.
      Nucleotide sequences in the yeast alanine transfer ribonucleic acid.
      ) isolated tRNAs and determined the first tRNA sequence. The first codons were mapped to amino acids using repeating polyribonucleotides as templates for protein synthesis (
      • Gardner R.S.
      • Wahba A.J.
      • Basilio C.
      • Miller R.S.
      • Lengyel P.
      • Speyer J.F.
      Synthetic polynucleotides and the amino acid code. VII.
      ,
      • Nishimura S.
      • Jones D.S.
      • Khorana H.G.
      Studies on polynucleotides. 48. The in vitro synthesis of a co-polypeptide containing two amino acids in alternating sequence dependent upon a DNA-like polymer containing two nucleotides in alternating sequence.
      ). The laboratories of Nirenberg et al. (
      • Nirenberg M.
      • Leder P.
      • Bernfield M.
      • Brimacombe R.
      • Trupin J.
      • Rottman F.
      • O'Neal C.
      RNA codewords and protein synthesis, VII. On the general nature of the RNA code.
      ) and Khorana et al. (
      • Söll D.
      • Ohtsuka E.
      • Jones D.S.
      • Lohrmann R.
      • Hayatsu H.
      • Nishimura S.
      • Khorana H.G.
      Studies on polynucleotides, XLIX. Stimulation of the binding of aminoacyl-sRNA’s to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids.
      ) then raced to solve the complete codon catalogue using a technique that monitored the binding of radiolabeled aminoacyl-tRNAs to ribosomes separately prepared with each of the possible 64 trinucleotide codons. These efforts established the standard genetic code table for which Holley, Khorana, and Nirenberg were awarded the Nobel prize in Physiology or Medicine in 1968.
      Fascinatingly, by the time the prize was awarded, exceptions to the genetic code had already been identified. In 1966, Yanofsky and co-workers (
      • Carbon J.
      • Berg P.
      • Yanofsky C.
      Missense suppression due to a genetically altered tRNA.
      ) demonstrated tRNA mutants enabled missense suppression or amino acid mis-incorporation using a defective tryptophan synthase A gene in Escherichia coli. Early in 1968, Atkins (
      • Riyasaty S.
      • Atkins J.F.
      External suppression of a frameshift mutant in Salmonella.
      ) identified the first exception to triplet decoding with the discovery of frame-shifting in Salmonella typhimurium. Indeed, although the genetic code is nearly universal in the living world, several exceptions to the standard code occur in diverse organisms (
      • Ling J.
      • O’Donoghue P.
      • Söll D.
      Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology.
      ). Genome sequences and genetic and biochemical data reveal that in organisms from microbes to humans, codons can be ambiguously decoded (
      • Carbon J.
      • Berg P.
      • Yanofsky C.
      Missense suppression due to a genetically altered tRNA.
      ,
      • Bezerra A.R.
      • Simões J.
      • Lee W.
      • Rung J.
      • Weil T.
      • Gut I.G.
      • Gut M.
      • Bayés M.
      • Rizzetto L.
      • Cavalieri D.
      • Giovannini G.
      • Bozza S.
      • Romani L.
      • Kapushesky M.
      • Moura G.R.
      • Santos M.A.
      Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification.
      ,
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ), reassigned (
      • Barrell B.G.
      • Bankier A.T.
      • Drouin J.
      A different genetic code in human mitochondria.
      ,
      • Campbell J.H.
      • O’Donoghue P.
      • Campbell A.G.
      • Schwientek P.
      • Sczyrba A.
      • Woyke T.
      • Söll D.
      • Podar M.
      UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota.
      ), or site-specifically recoded (
      • Böck A.
      • Stadtman T.C.
      Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism.
      ,
      • Berry M.J.
      • Martin 3rd., G.W.
      • Tujebajeva R.
      • Grundner-Culemann E.
      • Mansell J.B.
      • Morozova N.
      • Harney J.W.
      Selenocysteine insertion sequence element characterization and selenoprotein expression.
      • Bröcker M.J.
      • Ho J.M.
      • Church G.M.
      • Söll D.
      • O’Donoghue P.
      Recoding the genetic code with selenocysteine.
      ) to incorporate unexpected amino acids or amino acids beyond the standard set of 20.
      Given the importance of cytosolic tRNAs to facilitate accurate synthesis of the proteome, surprisingly few examples have linked a cytosolic tRNA mutation to human disease thus far. Yet, recent examples directly connecting cytosolic tRNA mutations to disease in humans (
      • Schoenmakers E.
      • Carlson B.
      • Agostini M.
      • Moran C.
      • Rajanayagam O.
      • Bochukova E.
      • Tobe R.
      • Peat R.
      • Gevers E.
      • Muntoni F.
      • Guicheney P.
      • Schoenmakers N.
      • Farooqi S.
      • Lyons G.
      • Hatfield D.
      • Chatterjee K.
      Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.
      ) and separately to neurodegeneration (
      • Kapur M.
      • Ackerman S.L.
      mRNA translation gone awry: translation fidelity and neurological disease.
      ,
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ) and cancer (
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      ) in mice suggest that cytosolic tRNA variants play a greater role in disease than previously imagined. It is possible that significant changes to tRNA function are not usually tolerated in the genome or that defective tRNA alleles may be genetically buffered by multiple copies of each iso-decoder. Nevertheless, two empirical observations suggest tRNAs have a larger role in disease than previously recognized: (i) the unexpectedly large number of tRNA variants in the human population (Table 1, Table 23 and Table S1), and (ii) the fact that even a single nucleotide change in a single tRNA gene can cause mistranslation or stall translation leading to molecular and cellular defects (
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ,
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ,
      • Berg M.D.
      • Hoffman K.S.
      • Genereaux J.
      • Mian S.
      • Trussler R.S.
      • Haniford D.B.
      • O'Donoghue P.
      • Brandl C.J.
      Evolving mistranslating tRNAs through a phenotypically ambivalent intermediate in Saccharomyces cerevisiae.
      ). The majority of research connecting human tRNA functions to disease is focused on mutations in aminoacyl-tRNA synthetases (AARSs)
      The abbreviations used are: AARS
      aminoacyl-tRNA synthetase
      SNP
      single nucleotide polymorphism
      TAM
      transcription-associated mutagenesis
      CFTR
      cystic fibrosis transmembrane conductance regulator
      mcm5U
      5-methoxycarbonylmethyluridine
      mcm5Um
      5-methoxycarbonylmethyl-2′-O-methyluridine
      HIF
      hypoxia-inducible factor
      GtRNAdb
      Genomic tRNA database
      pol III
      polymerase III
      HD
      Huntington's disease.
      (
      • Meyer-Schuman R.
      • Antonellis A.
      Emerging mechanisms of aminoacyl-tRNA synthetase mutations in recessive and dominant human disease.
      ), on proteins that modify nucleotides in cytosolic tRNAs (
      • Bednářová A.
      • Hanna M.
      • Durham I.
      • VanCleave T.
      • England A.
      • Chaudhuri A.
      • Krishnan N.
      Lost in translation: defects in transfer RNA modifications and neurological disorders.
      ,
      • Torres A.G.
      • Batlle E.
      • Ribas de Pouplana L.
      Role of tRNA modifications in human diseases.
      ), or on the smaller pool of mitochondrial tRNAs (
      • Kirchner S.
      • Ignatova Z.
      Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.
      ,
      • Abbott J.A.
      • Francklyn C.S.
      • Robey-Bond S.M.
      Transfer RNA and human disease.
      • Yasukawa T.
      • Suzuki T.
      • Ishii N.
      • Ohta S.
      • Watanabe K.
      Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease.
      ). Two major reasons for this are a relative lack of available sequence data for cytosolic tRNAs, and a long-held assumption that excessive tRNA copy number should “buffer” potential phenotypes resulting from a single mutant.
      Table 1Human tRNA anticodon variants
      tRNA geneVariantMAF (%)
      Data are from the 1000 Genomes Project (127, 128).
      Variant count
      Data are from the 1000 Genomes Project (127, 128).
      MAF (%)
      Data are from the TOPMED sequencing project (128, 129).
      Variant count
      Data are from the TOPMED sequencing project (128, 129).
      tRNA score
      tRNA score was calculated using tRNA-Scan SE (128).
      Codon identitytRNA identityExpression
      ARM
      ARM indicates ARM-seq data suggesting expression (128, 130).
      CHIP
      CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (46, 129, 131–133).
      Ala-AGC-2–2G35A0.02184.7ValAla++
      Ala-AGC-6–1G35C6.553286.47813074.1GlyAla++
      Ala-AGC-15–1C36T0.0420.034256.7ThrAla+
      Ala-AGC-16–1G35A2.2112.126953.1ValAla+
      Ala-CGC-1–1G35T0.0008179.7GluAla++
      Ala-TGC-1–1G35A80.5ValAla++
      Leu-CAA-3–1A35C0.0008177.3TrpLeu++
      Ser-AGA-2–2G35A0.020.003489.6PheSer++
      Ser-AGA-2–3G35A1.82911.87234789.6PheSer++
      Ser-AGA-2–4G35C0.003489.6CysSer++
      Ser-AGA-2–5_35T0.0008189.6++
      Ser-CGA-2–1_36T0.033494+
      Ser-TGA-2–1G35A0.0420.022290.4LeuSer++
      Ser-TGA-3–1_36T0.0008189.7++
      a Data are from the 1000 Genomes Project (
      • Parisien M.
      • Wang X.
      • Pan T.
      Diversity of human tRNA genes from the 1000-genomes project.
      ,
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ).
      b Data are from the TOPMED sequencing project (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ).
      c tRNA score was calculated using tRNA-Scan SE (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ).
      d ARM indicates ARM-seq data suggesting expression (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Hrabeta-Robinson E.
      • Marcus E.
      • Cozen A.E.
      • Phizicky E.M.
      • Lowe T.M.
      High-throughput small RNA sequencing enhanced by AlkB-facilitated RNA de-methylation (ARM-Seq).
      ).
      e CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (
      • Canella D.
      • Praz V.
      • Reina J.H.
      • Cousin P.
      • Hernandez N.
      Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ,
      • Gerstein M.B.
      • Kundaje A.
      • Hariharan M.
      • Landt S.G.
      • Yan K.K.
      • Cheng C.
      • Mu X.J.
      • Khurana E.
      • Rozowsky J.
      • Alexander R.
      • Min R.
      • Alves P.
      • Abyzov A.
      • Addleman N.
      • Bhardwaj N.
      • et al.
      Architecture of the human regulatory network derived from ENCODE data.
      ,
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.
      • Whitfield T.W.
      • Greven M.C.
      • Pierce B.G.
      • Dong X.
      • Kundaje A.
      • Cheng Y.
      • Rando O.J.
      • Birney E.
      • Myers R.M.
      • Noble W.S.
      • Snyder M.
      • Weng Z.
      Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.Y.
      • Greven M.C.
      • Kim B.H.
      • Moore J.
      • Pierce B.G.
      • Dong X.
      • Virgil D.
      • Birney E.
      • Hung J.H.
      • Weng Z.
      Factorbook.org: a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium.
      ).
      Table 2Human tRNA variants that introduce a G3:U70 base pair
      tRNA geneVariantMAF (%)
      Data are from the 1000 Genomes Project (127, 128).
      Variant count
      Data are from the 1000 Genomes Project (127, 128).
      MAF (%)
      Data are from the TOPMED sequencing project (128, 129).
      Variant count
      Data are from the TOPMED sequencing project (128, 129).
      tRNA score
      tRNA score was calculated using tRNA-Scan SE (128).
      Expression
      ARM
      ARM indicates ARM-seq data suggesting expression (128, 130).
      CHIP
      CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (46, 129, 131–133).
      Arg-ACG-1–3C70U0.002268++
      Cys-GCA-2–3C70U0.022982++
      Cys-GCA-1–1C70U0.0210.078684+
      Cys-GCA-17–1C70U0.002271++
      Cys-GCA-12–1C70U0.0210.011672++
      Gly-CCC-2–1G70U0.056475++
      Gly-GCC-2–4A3G0.0840.056381+
      Gly-GCC-2–5A3G0.0210.005681+
      Gly-GCC-2–1A3G0.0210.022081++
      Gly-GCC-2–3A3G81++
      Gly-GCC-1–5A3G1.2611.3163381
      Gly-GCC-5–1A3G0.0210.112955
      Gly-TCC-2–6C70U0.03140.0911174++
      Ser-AGA-2–6A3G0.011290++
      a Data are from the 1000 Genomes Project (
      • Parisien M.
      • Wang X.
      • Pan T.
      Diversity of human tRNA genes from the 1000-genomes project.
      ,
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ).
      b Data are from the TOPMED sequencing project (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ).
      c tRNA score was calculated using tRNA-Scan SE (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ).
      d ARM indicates ARM-seq data suggesting expression (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Hrabeta-Robinson E.
      • Marcus E.
      • Cozen A.E.
      • Phizicky E.M.
      • Lowe T.M.
      High-throughput small RNA sequencing enhanced by AlkB-facilitated RNA de-methylation (ARM-Seq).
      ).
      e CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (
      • Canella D.
      • Praz V.
      • Reina J.H.
      • Cousin P.
      • Hernandez N.
      Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ,
      • Gerstein M.B.
      • Kundaje A.
      • Hariharan M.
      • Landt S.G.
      • Yan K.K.
      • Cheng C.
      • Mu X.J.
      • Khurana E.
      • Rozowsky J.
      • Alexander R.
      • Min R.
      • Alves P.
      • Abyzov A.
      • Addleman N.
      • Bhardwaj N.
      • et al.
      Architecture of the human regulatory network derived from ENCODE data.
      ,
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.
      • Whitfield T.W.
      • Greven M.C.
      • Pierce B.G.
      • Dong X.
      • Kundaje A.
      • Cheng Y.
      • Rando O.J.
      • Birney E.
      • Myers R.M.
      • Noble W.S.
      • Snyder M.
      • Weng Z.
      Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.Y.
      • Greven M.C.
      • Kim B.H.
      • Moore J.
      • Pierce B.G.
      • Dong X.
      • Virgil D.
      • Birney E.
      • Hung J.H.
      • Weng Z.
      Factorbook.org: a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium.
      ).
      Table 3Human tRNAArg C50T variants
      tRNA geneSNP IDMAF (%)
      Data are from the 1000 genomes project.
      Variant count
      Data are from the 1000 genomes project.
      MAF (%)
      Data are from the TOPMED sequencing project (128, 129).
      Variant count
      Data are from the TOPMED sequencing project (128, 129).
      tRNA score
      tRNA score was calculated using tRNA-Scan SE (128).
      Expression
      ARM
      ARM indicates ARM-seq data suggesting expression (128, 130).
      CHIP
      CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (46, 129, 131–133).
      Arg-ACG-1–1rs69395402.421212.2276567.6++
      Arg-ACG-1–2rs1861041070.0210.011467.6++
      Arg-TCG-6–1rs11317004316.683120.224,37453.7+
      Arg-TCT-5–1rs1433342720.8410.671161.4+
      a Data are from the 1000 genomes project.
      b Data are from the TOPMED sequencing project (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ).
      c tRNA score was calculated using tRNA-Scan SE (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ).
      d ARM indicates ARM-seq data suggesting expression (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ,
      • Hrabeta-Robinson E.
      • Marcus E.
      • Cozen A.E.
      • Phizicky E.M.
      • Lowe T.M.
      High-throughput small RNA sequencing enhanced by AlkB-facilitated RNA de-methylation (ARM-Seq).
      ).
      e CHIP indicates CHIP-seq hits for at least 3 of 4 core transcription proteins (RPC155, POLR3G, BRF1, and BDP1) (
      • Canella D.
      • Praz V.
      • Reina J.H.
      • Cousin P.
      • Hernandez N.
      Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells.
      ,
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • Raney B.J.
      • Lee C.M.
      • Lee B.T.
      • Karolchik D.
      • Hinrichs A.S.
      • Haeussler M.
      • Guruvadoo L.
      • Navarro Gonzalez J.
      • et al.
      The UCSC genome browser database: 2018 update.
      ,
      • Gerstein M.B.
      • Kundaje A.
      • Hariharan M.
      • Landt S.G.
      • Yan K.K.
      • Cheng C.
      • Mu X.J.
      • Khurana E.
      • Rozowsky J.
      • Alexander R.
      • Min R.
      • Alves P.
      • Abyzov A.
      • Addleman N.
      • Bhardwaj N.
      • et al.
      Architecture of the human regulatory network derived from ENCODE data.
      ,
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.
      • Whitfield T.W.
      • Greven M.C.
      • Pierce B.G.
      • Dong X.
      • Kundaje A.
      • Cheng Y.
      • Rando O.J.
      • Birney E.
      • Myers R.M.
      • Noble W.S.
      • Snyder M.
      • Weng Z.
      Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.
      • Wang J.
      • Zhuang J.
      • Iyer S.
      • Lin X.Y.
      • Greven M.C.
      • Kim B.H.
      • Moore J.
      • Pierce B.G.
      • Dong X.
      • Virgil D.
      • Birney E.
      • Hung J.H.
      • Weng Z.
      Factorbook.org: a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium.
      ).
      In this review, we outline the complexity of cytosolic tRNA function and regulation in eukaryotic cells. We then summarize recent studies demonstrating examples of single nucleotide tRNA variants that elicit significant levels of amino acid mis-incorporation, which can be surprisingly well-tolerated in eukaryotic cells. Using data from the 1000 Genomes Project, we analyzed the location and frequency of naturally occurring human tRNA variants. These data reveal an abundance of mistranslating tRNAs in the human population. Finally, we summarize recent evidence linking tRNA mutations and de-regulated tRNA expression and nucleotide modification to disease in humans and model systems. Some of the studies point to the idea that tRNA mutations, which are otherwise tolerated or benign, contribute to disease in the context of other coincidental cellular defects.

      tRNA function and regulation

      The role of tRNA in decoding the genetic code

      tRNAs are best known for their role in translation of RNA messages into proteins. tRNAs are relatively small RNA molecules, typically consisting of 76–90 nucleotides, and fold into a conserved three-dimensional structure in the shape of an upside-down L (Fig. 1A). The anticodon resides at the long end of the L-shape and binds to cognate codons in the messenger RNA (mRNA) on the ribosome. On the opposite end of the tRNA, the amino acid is ligated to the 3′-terminal adenosine residue in the acceptor stem. Accurate tRNA aminoacylation and high-fidelity decoding of codons on the ribosome are key determinants to accurate protein production.
      Figure thumbnail gr1
      Figure 1tRNA structure. A, tRNAs fold into an L-shaped three-dimensional structure with extensive intramolecular base-pairing. This tRNAPhe structure (Protein Data Bank code 1OB5) (
      • Parmeggiani A.
      • Krab I.M.
      • Watanabe T.
      • Nielsen R.C.
      • Dahlberg C.
      • Nyborg J.
      • Nissen P.
      Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics.
      ) is aminoacylated with phenylalanine (AA). B, in a two-dimensional representation, tRNA resembles a cloverleaf. In both diagrams, the tRNA is colored by structural elements: acceptor stem (red), dihydrouridine (D)-arm (green), anticodon stem (cyan), anticodon (bases 34–36 in purple), variable loop (yellow), TΨC (T) arm (navy), and the conserved CCA-3′ end (white). A schematic mRNA is shown below the tRNA diagram to indicate the tRNA nucleotides that base pair with each codon position.
      Codon recognition is determined by the tRNA anticodon, which base pair with tri-nucleotide codons in mRNAs during protein synthesis (Fig. 1B). The essential interaction between codon and anticodon is established not only by Watson-Crick base pairing, but also by nucleotide modifications in tRNAs (
      • Agris P.F.
      • Vendeix F.A.
      • Graham W.D.
      tRNA's wobble decoding of the genome: 40 years of modification.
      ,
      • Joshi K.
      • Bhatt M.J.
      • Farabaugh P.J.
      Codon-specific effects of tRNA anticodon loop modifications on translational misreading errors in the yeast Saccharomyces cerevisiae.
      ), competition between cognate and near-cognate decoding (
      • Pavlov M.Y.
      • Ehrenberg M.
      Substrate-induced formation of ribosomal decoding center for accurate and rapid genetic code translation.
      ,
      • O’Donoghue P.
      • Prat L.
      • Heinemann I.U.
      • Ling J.
      • Odoi K.
      • Liu W.R.
      • Söll D.
      Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion.
      ), and wobble decoding. Generally, the first two positions of a codon form Watson-Crick pairs with the tRNA, whereas the third position is more flexible (
      • Agris P.F.
      • Vendeix F.A.
      • Graham W.D.
      tRNA's wobble decoding of the genome: 40 years of modification.
      ). In back-to-back publications, Crick hypothesized (
      • Crick F.H.
      Codon–anticodon pairing: the wobble hypothesis.
      ) what Söll et al. (
      • Söll D.
      • Jones D.S.
      • Ohtsuka E.
      • Faulkner R.D.
      • Lohrmann R.
      • Hayatsu H.
      • Khorana H.G.
      Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique.
      ) determined experimentally that the third position of a codon can involve G:U or U:G wobble pairing with the 1st position of the anticodon at tRNA nucleotide 34. Indeed, the initial discoveries also included examples of extended wobble decoding in yeast arginine and alanine tRNAs that read codons ending in U, C, or A (
      • Söll D.
      • Jones D.S.
      • Ohtsuka E.
      • Faulkner R.D.
      • Lohrmann R.
      • Hayatsu H.
      • Khorana H.G.
      Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique.
      ). Extended wobble decoding is facilitated by post-transcriptional tRNA modification, where adenosine residues at position 34 are modified to inosine, which pairs with U, C, or A in the third codon position (
      • Agris P.F.
      • Vendeix F.A.
      • Graham W.D.
      tRNA's wobble decoding of the genome: 40 years of modification.
      ). Additional nucleotide modifications in the anticodon loop (particularly at positions 34 and 37) also impact translation fidelity and reading-frame maintenance (
      • Agris P.F.
      • Narendran A.
      • Sarachan K.
      • Väre V.Y.P.
      • Eruysal E.
      The importance of being modified: the role of RNA modifications in translational fidelity.
      ,
      • Gustilo E.M.
      • Vendeix F.A.
      • Agris P.F.
      tRNA's modifications bring order to gene expression.
      ). For example, in yeast, a 5-methoxycarbonylmethyl-2-thiouridine modification at anticodon base U34 represses +1 frame-shifting. Lack of the modification or hypo-modification at this site in a variety of tRNAs leads to 1.5–3.0-fold increases in ribosomal frame-shifting (
      • Tükenmez H.
      • Xu H.
      • Esberg A.
      • Byström A.S.
      The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes.
      ). Similarly, absence of the modified base N6-threonylcarbamoyladenosine at the anticodon adjacent position 37 also increases frame-shifting in yeast by 2-fold (
      • Lin C.A.
      • Ellis S.R.
      • True H.L.
      The Sua5 protein is essential for normal translational regulation in yeast.
      ).
      The standard genetic code is composed of 61 sense codons that encode 20 amino acids and 3 stop codons (UGA, UAG, and UAA) that usually signal termination of protein synthesis. Because certain tRNAs decode up to three or four different codons, the theoretical minimum number of tRNAs for an organism to encode 20 amino acids is 32 (
      • Crick F.H.
      Codon–anticodon pairing: the wobble hypothesis.
      ). Söll et al. (
      • Söll D.
      • Jones D.S.
      • Ohtsuka E.
      • Faulkner R.D.
      • Lohrmann R.
      • Hayatsu H.
      • Khorana H.G.
      Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique.
      ) observed that the “minimum number of sRNA [tRNA] molecules required for recognition of all of the meaningful codons is relatively small, and this conclusion in turn raises the question of redundancy in the sRNA pool of a cell.” The question as to why organisms encode apparently redundant tRNA genes, which was raised the year after the code was solved, is still unanswered today. As a result of the genome sequencing revolution, we know now that nature contains examples of organisms with tRNA gene complements that are well below and vastly greater than this apparent minimal requirement. There are examples of organelles (e.g. human mitochondria with 22 tRNA genes) and even parasitic microbes (e.g. Mycoplasma mobile with 28 tRNA genes (
      • Jaffe J.D.
      • Stange-Thomann N.
      • Smith C.
      • DeCaprio D.
      • Fisher S.
      • Butler J.
      • Calvo S.
      • Elkins T.
      • FitzGerald M.G.
      • Hafez N.
      • Kodira C.D.
      • Major J.
      • Wang S.
      • Wilkinson J.
      • Nicol R.
      • Nusbaum C.
      • et al.
      The complete genome and proteome of Mycoplasma mobile.
      )) with fewer than 32 tRNA genes. Their survival depends on importing the missing tRNAs from a different cellular compartment (
      • Chatterjee K.
      • Nostramo R.T.
      • Wan Y.
      • Hopper A.K.
      tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: location, location, location.
      ) or presumably a host cell.
      E. coli encodes 88 tRNA genes, whereas yeast has a small tRNAome for a eukaryote at 275 tRNA genes. Eukaryotes typically have hundreds of tRNA genes that display a general trend to increase in number and sequence diversity with the complexity of the organism (
      • Goodenbour J.M.
      • Pan T.
      Diversity of tRNA genes in eukaryotes.
      ). Unicellular protozoans encode near the theoretical minimum of tRNA genes, such as the malaria parasite Plasmodium falciparum, which has only 35 tRNA genes (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
      ). P. falciparum was recently found to import additional tRNAs from its host (
      • Bour T.
      • Mahmoudi N.
      • Kapps D.
      • Thiberge S.
      • Bargieri D.
      • Ménard R.
      • Frugier M.
      Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites.
      ). Some species of fish have astoundingly high tRNA gene numbers (Table S2), such as the elephant shark (Callorhinchus milii), which encodes 13,724 tRNAs (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
      ). As exemplified in a phylogenetic comparison of yeast and human alanine tRNAs (Fig. 2), the sequence variations among tRNA iso-acceptors appear to increase with complexity as well. Yeast has 16 tRNAAla iso-acceptors, including 11 identical genes with the AGC anticodon and 5 identical genes with the TGC anticodon. In contrast, humans encode 45 tRNAAla iso-acceptors with markedly greater sequence diversity than their yeast counterparts, including examples with CGC anticodons not seen in yeast (Fig. 2).
      Figure thumbnail gr2
      Figure 2Phylogenetic relationships of human and yeast tRNAAla. The tree is based on an alignment of all known human and yeast tRNAAla iso-acceptors. The vastly expanded number and greater diversity of human tRNAAla genes compared with their yeast counterparts are evident. The tRNAs are labeled according to gene names in the genomic tRNA database (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
      ), which include anticodon sequence followed by a numbering system where the first number indicates similar sequences, and the second number indicates a gene copy identifier. The human reference genome contains a misannotated tRNAAla, which was used to root the tree. This gene, tRNAAla-GGC-19-3, is a tRNAThr with a mutation (T36C) endowing the tRNA with an alanine anticodon. This is the only example of the alanine GGC anticodon in humans. Scale bar indicates the number of nucleotide changes per site in the tRNA sequences. The tree was calculated similarly as before (
      • Hohn M.J.
      • Park H.S.
      • O’Donoghue P.
      • Schnitzbauer M.
      • Söll D.
      Emergence of the universal genetic code imprinted in an RNA record.
      ). Briefly, a starting tree computed in MultiSeq 2.0 (
      • Roberts E.
      • Eargle J.
      • Wright D.
      • Luthey-Schulten Z.
      MultiSeq: unifying sequence and structure data for evolutionary analysis.
      ) was optimized to identify the maximum likelihood tree using PhyML 3.1 (
      • Guindon S.
      • Dufayard J.F.
      • Lefort V.
      • Anisimova M.
      • Hordijk W.
      • Gascuel O.
      New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0.
      ). Statistical branch support (out of 100) was calculated based on an approximate likelihood ratio test method (
      • Shimodaira H.
      • Hasegawa M.
      Multiple comparisons of log-likelihoods with applications to phylogenetic inference.
      ) as implemented in PhyML.

      tRNA regulation in human cells

      The number of expressed tRNA genes in human cells is not well defined. Of the 610 tRNA genes in humans, the genomic tRNA database predicts 417 genes in their high-confidence set, indicating the tRNA is likely to function in protein synthesis (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
      ). Comprehensive profiling of RNAs in human serum suggests 411 expressed tRNA genes (
      • Umu S.U.
      • Langseth H.
      • Bucher-Johannessen C.
      • Fromm B.
      • Keller A.
      • Meese E.
      • Lauritzen M.
      • Leithaug M.
      • Lyle R.
      • Rounge T.B.
      A comprehensive profile of circulating RNAs in human serum.
      ). According to CHIP-seq analysis of RNA polymerase III and transcription factor occupancy, ∼350 tRNA genes are actively transcribed in a single human cell line (IMR90hTert) (
      • Canella D.
      • Praz V.
      • Reina J.H.
      • Cousin P.
      • Hernandez N.
      Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells.
      ). Gogakos et al. (
      • Gogakos T.
      • Brown M.
      • Garzia A.
      • Meyer C.
      • Hafner M.
      • Tuschl T.
      Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP.
      ) reported the expression of 288–349 tRNAs in HEK 293 cells based on two different RNA-Seq methods. Together, the data suggest 300–400 tRNA genes are expressed in any individual human cell.
      The degree to which each human tRNA contributes to protein synthesis has not been determined, but evidence that cells regulate tRNA expression to control protein production is emerging. First, expression of individual tRNA genes varies between tissues (
      • Dittmar K.A.
      • Goodenbour J.M.
      • Pan T.
      Tissue-specific differences in human transfer RNA expression.
      ,
      • Melé M.
      • Ferreira P.G.
      • Reverter F.
      • DeLuca D.S.
      • Monlong J.
      • Sammeth M.
      • Young T.R.
      • Goldmann J.M.
      • Pervouchine D.D.
      • Sullivan T.J.
      • Johnson R.
      • Segrè A.V.
      • Djebali S.
      • Niarchou A.
      • et al.
      GTEx Consortium
      Human genomics. The human transcriptome across tissues and individuals.
      ). Furthermore, the steady-state level of different tRNAs correlates with the expression of matched-codon biased mRNA transcripts (
      • Dittmar K.A.
      • Goodenbour J.M.
      • Pan T.
      Tissue-specific differences in human transfer RNA expression.
      ). The observation suggests that cells can fine-tune tRNA expression profiles to match codon usage in expressed mRNAs. Indeed, the fact that efficient protein expression requires tRNA levels and decoding capacity matches the distribution of codons in mRNA is well known. Multiple E. coli strains and bioinformatic tools for codon adaptation were developed based on this principle to enhance the production of eukaryotic and other recombinant proteins in bacteria (
      • Grote A.
      • Hiller K.
      • Scheer M.
      • Münch R.
      • Nörtemann B.
      • Hempel D.C.
      • Jahn D.
      JCat: a novel tool to adapt codon usage of a target gene to its potential expression host.
      ).
      Once transcribed, tRNAs are processed via the removal and addition of nucleotides to produce a mature tRNA. Introns, 5′-leader, and 3′-trailer sequences in the original transcript are removed (
      • Betat H.
      • Long Y.
      • Jackman J.E.
      • Mörl M.
      From end to end: tRNA editing at 5′- and 3′-terminal positions.
      ). Next, the CCA-adding enzyme elongates the pre-tRNA with the conserved CCA 3′-end. CCA-adding enzymes can append a second CCA to certain tRNAs with mismatches or excessive G:U pairs in their acceptor stems (
      • Wilusz J.E.
      Controlling translation via modulation of tRNA levels: controlling translation via modulation of tRNA levels.
      ). The double CCA addition primes tRNAs for exonucleolytic digestion via the rapid tRNA decay pathway (
      • Wilusz J.E.
      • Whipple J.M.
      • Phizicky E.M.
      • Sharp P.A.
      tRNAs marked with CCACCA are targeted for degradation.
      ). The human CCA-adding enzyme (TRNT1) is implicated in disease. Complete loss-of-function in TRNT1 is embryonic lethal, and partial loss-of-function mutations cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (
      • Chakraborty P.K.
      • Schmitz-Abe K.
      • Kennedy E.K.
      • Mamady H.
      • Naas T.
      • Durie D.
      • Campagna D.R.
      • Lau A.
      • Sendamarai A.K.
      • Wiseman D.H.
      • May A.
      • Jolles S.
      • Connor P.
      • Powell C.
      • Heeney M.M.
      • et al.
      Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).
      ).
      tRNAs are further processed with a variety of post-transcriptional nucleotide modifications, including 2′ O-methylation of the ribose, N-acetylation and N-methylation of the nucleotide bases, as well as more complex modifications that form bases such as wybutosine (
      • Lorenz C.
      • Lunse C.E.
      • Morl M.
      tRNA modifications: impact on structure and thermal adaptation.
      ).
      In eukaryotes, tRNAHis is post-transcriptionally edited to add an extra guanine (G−1) to the 5′-end of the tRNA, a unique feature recognized and required by the cognate histidyl-tRNA synthetase (
      • Heinemann I.U.
      • Nakamura A.
      • O’Donoghue P.
      • Eiler D.
      • Söll D.
      tRNAHis-guanylyltransferase establishes tRNAHis identity.
      ). Depleting the tRNAHis guanylyltransferase that catalyzes G−1 addition leads to accumulation of un-aminoacylated and un-guanylated yet hyper-methylated tRNAHis in the nucleus (
      • Gu W.
      • Hurto R.L.
      • Hopper A.K.
      • Grayhack E.J.
      • Phizicky E.M.
      Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C.
      ). Subsequent studies point to tRNAHis m5C hypermethylation as a response to growth arrest in Saccharomyces cerevisiae, although the significance of the increase in m5C methylation is yet unclear (
      • Preston M.A.
      • D'Silva S.
      • Kon Y.
      • Phizicky E.M.
      tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae.
      ). Monomethylation of the 5′-monophosphate of tRNAHis by bicoid-interacting 3 domain containing RNA methyltransferase (BCDIN3D) is thought to protect tRNAHis from degradation. BCDIN3D is overexpressed in breast cancer cells, and monomethylated tRNAHis is more abundant in breast cancer cells, yet the overall level of tRNAHis is not impacted (
      • Martinez A.
      • Yamashita S.
      • Nagaike T.
      • Sakaguchi Y.
      • Suzuki T.
      • Tomita K.
      Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA.
      ). It is thought that monomethylation contributes to the formation of tRNAHis-derived fragments in breast cancer cells, which in turn regulate tumorigenic genes involved in breast cancers (
      • Tomita K.
      • Liu Y.
      Human BCDIN3D is a cytoplasmic tRNA(His)-specific 5′-monophosphate methyltransferase.
      ).
      In fact, tRNAs are the most frequently modified noncoding RNA known, containing an average of 13 modifications per molecule (
      • Saikia M.
      • Fu Y.
      • Pavon-Eternod M.
      • He C.
      • Pan T.
      Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs.
      ). The combined number of expressed tRNA genes and their multiple modification states imply the existence of a large combinatorial number of tRNA microspecies in the human cell (
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ). Because tRNA modifications are important for translation fidelity and reading frame maintenance (
      • Crick F.H.
      Codon–anticodon pairing: the wobble hypothesis.
      ,
      • Agris P.F.
      • Narendran A.
      • Sarachan K.
      • Väre V.Y.P.
      • Eruysal E.
      The importance of being modified: the role of RNA modifications in translational fidelity.
      ,
      • Gustilo E.M.
      • Vendeix F.A.
      • Agris P.F.
      tRNA's modifications bring order to gene expression.
      ,
      • Towns W.L.
      • Begley T.J.
      Transfer RNA methyltransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health.
      ), these microspecies have the potential to impact cellular function and disease. Modifications are also essential for regulating tRNA turnover (
      • Hopper A.K.
      Transfer RNA post-transcriptional processing, turnover, and subcellular dynamics in the yeast Saccharomyces cerevisiae.
      ) and for proper structure, folding, and stability of the tRNA (
      • Lorenz C.
      • Lunse C.E.
      • Morl M.
      tRNA modifications: impact on structure and thermal adaptation.
      ). Indeed, many tRNA-modifying enzymes are already linked to disease (
      • Torres A.G.
      • Batlle E.
      • Ribas de Pouplana L.
      Role of tRNA modifications in human diseases.
      ). As described below, tRNA modification can also dynamically up- or down-regulate sets of tRNAs (
      • Dedon P.C.
      • Begley T.J.
      A system of RNA modifications and biased codon use controls cellular stress response at the level of translation.
      ,
      • Endres L.
      • Dedon P.C.
      • Begley T.J.
      Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses.
      • Rapino F.
      • Delaunay S.
      • Rambow F.
      • Zhou Z.
      • Tharun L.
      • De Tullio P.
      • Sin O.
      • Shostak K.
      • Schmitz S.
      • Piepers J.
      • Ghesquière B.
      • Karim L.
      • Charloteaux B.
      • Jamart D.
      • Florin A.
      • et al.
      Codon-specific translation reprogramming promotes resistance to targeted therapy.
      ).
      The mature and active tRNA is a substrate for amino acid ligation catalyzed by the AARS enzymes in an ATP-dependent reaction (
      • Ibba M.
      • Soll D.
      Aminoacyl-tRNA synthesis.
      ). Each AARS enzyme has specificity both for an amino acid and a distinct set of cognate tRNA iso-acceptors. Amino acids are ligated to the 3′-end of tRNAs, requiring the presence of a CCA 3′-tail. To ensure tRNA recognition fidelity, AARSs make essential contacts with nucleotides in their cognate tRNAs, called identity elements (
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ). Aminoacyl-tRNAs are then substrates for protein synthesis. The likelihood that a given aminoacyl-tRNA acts in translation depends upon many factors, including the stability of the tRNA, the number of aminoacylated-tRNAs competing for the same codon, and the expression of mRNAs containing codons read by the tRNA. tRNAs unfit for translation are degraded by the rapid tRNA decay pathway (
      • Payea M.J.
      • Sloma M.F.
      • Kon Y.
      • Young D.L.
      • Guy M.P.
      • Zhang X.
      • De Zoysa T.
      • Fields S.
      • Mathews D.H.
      • Phizicky E.M.
      Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA.
      ). Cytoplasmic tRNA levels are also regulated by export processes to other cellular compartments, including into mitochondria (
      • Kessler A.C.
      • Silveira d'Almeida G.
      • Alfonzo J.D.
      The role of intracellular compartmentalization on tRNA processing and modification.
      ), or retrograde transport into the nucleus (
      • Hopper A.K.
      • Pai D.A.
      • Engelke D.R.
      Cellular dynamics of tRNAs and their genes.
      ). As reviewed elsewhere (
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ), tRNAs perform additional functions outside of translation, either as whole tRNAs (
      • Kwon N.H.
      • Lee M.R.
      • Kong J.
      • Park S.K.
      • Hwang B.J.
      • Kim B.G.
      • Lee E.-S.
      • Moon H.-G.
      • Kim S.
      Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation.
      ) or tRNA-derived fragments (
      • Lee Y.S.
      • Shibata Y.
      • Malhotra A.
      • Dutta A.
      A novel class of small RNAs: tRNA-derived RNA fragments (tRFs).
      ).

      Phenotypes of mistranslating cells

      Mistranslation occurs in all cells (
      • Schwartz M.H.
      • Pan T.
      Function and origin of mistranslation in distinct cellular contexts.
      ) as a result of multiple different mechanisms. Considering the small size, multitude of protein partners, and essential cellular role of tRNAs, single nucleotide changes can have a profound impact on their function and on the efficiency and fidelity of protein synthesis (
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      ,
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ,
      • Berg M.D.
      • Hoffman K.S.
      • Genereaux J.
      • Mian S.
      • Trussler R.S.
      • Haniford D.B.
      • O'Donoghue P.
      • Brandl C.J.
      Evolving mistranslating tRNAs through a phenotypically ambivalent intermediate in Saccharomyces cerevisiae.
      ,
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ). Proteins encoded by mRNAs containing rare codons or strongly biased codon compositions are most susceptible to the effects of tRNA variants. Loss-of-function mutations in tRNAs can cause ribosome stalling to de-regulate protein synthesis, whereas gain-of-function mutations in tRNAs can lead to mis-aminoacylation and mistranslation (
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      ,
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ).
      Mistranslating tRNAs can arise from surprisingly minor changes to the nucleotide sequence. Although many tRNAs harbor major identity determinants in their anticodon, coupling aminoacylation fidelity to codon assignment, alanyl-, leucyl-, and seryl-tRNA synthetases do not recognize the anticodon nucleotides on their cognate tRNAs. Anticodon mutations in these tRNAs often elicit amino acid mis-incorporation (
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ). The accumulation of highly active tRNASer anticodon mutants is toxic to yeast cells, causing proteome-wide mistranslation (
      • Berg M.D.
      • Hoffman K.S.
      • Genereaux J.
      • Mian S.
      • Trussler R.S.
      • Haniford D.B.
      • O'Donoghue P.
      • Brandl C.J.
      Evolving mistranslating tRNAs through a phenotypically ambivalent intermediate in Saccharomyces cerevisiae.
      ). In yeast, the degree of anticodon mutant toxicity varies, depending on competition with WT tRNAs, chemical properties of the amino acids, and tRNA modifications (
      • Zimmerman S.M.
      • Kon Y.
      • Hauke A.C.
      • Ruiz B.Y.
      • Fields S.
      • Phizicky E.M.
      Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.
      ).
      Santos et al. (
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      )analyzed tRNASer variants containing Ala or Leu anticodons in murine NIH 3T3 cells grown in culture and subsequently xenografted to live mice. As determined by mass spectrometry in tumor samples recovered from the mice, the rate of mistranslation increased by ∼2-fold in the cells expressing tRNASer containing an alanine anticodon, but only marginally in cells expressing tRNASer with a leucine anticodon. Mistranslation was not toxic to the NIH 3T3 cells when grown in culture as determined by cellular viability, necrosis, and proliferation assays, indicating that increased cytosolic tRNA-dependent mistranslation was initially well-tolerated. Interestingly, expressing the mistranslating tRNAs promoted the formation of foci in vitro, suggesting a link to tumorigenesis (
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      ). Briefly, foci formation occurs when cancer-like cells form dense clusters resembling early-stage tumors on a Petri dish (
      • Alvarez A.
      • Barisone G.A.
      • Diaz E.
      Focus formation: a cell-based assay to determine the oncogenic potential of a gene.
      ). Mistranslating tRNAs promoted the activation of the oncogenic factors protein kinase B (Akt) and p38 when cells were treated with tumor necrosis factor-α, to a greater extent than cells expressing the WT tRNAs. Furthermore, cells mistranslating alanine codons with serine promoted angiogenesis in a chick chorioallantoic membrane assay and were highly tumorigenic when introduced in mice. Compared with the parent cells in culture, expression of the mistranslating tRNAs increased ∼8-fold in cells recovered from mouse tumors. Although mistranslating tRNA variants had undetectable cytotoxicity in cells in culture, the mutant tRNAs exacerbated or accelerated cellular pathways to cancer in a mammalian model of disease (
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      ).
      Identity element mutations are another route to mistranslating tRNAs. The phenotypic consequences of a single tRNA variant of this type are the subject of a number of recent studies. AlaRS recognizes two critical identity determinants at the 3rd base pair in the acceptor stem (G3:U70) (
      • Hou Y.-M.
      • Francklyn C.
      • Schimmel P.
      Molecular dissection of a transfer RNA and the basis for its identity.
      ), and also aminoacylates tRNAs bearing the GU pair at the 4th acceptor stem base pair (
      • Zimmerman S.M.
      • Kon Y.
      • Hauke A.C.
      • Ruiz B.Y.
      • Fields S.
      • Phizicky E.M.
      Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.
      ). tRNA variants that convert nonalanine tRNAs to alanine accepting tRNAs by creating these identity elements are common in mammalian genomes (
      • Sun L.
      • Gomes A.C.
      • He W.
      • Zhou H.
      • Wang X.
      • Pan D.W.
      • Schimmel P.
      • Pan T.
      • Yang X.-L.
      Evolutionary gain of alanine mischarging to noncognate tRNAs with a G4:U69 base pair.
      ). Some human tRNACys and tRNAThr species with G4:U69 are natural alanine acceptors, and cysteine to alanine mistranslation was detected in HEK 293 cells (
      • Sun L.
      • Gomes A.C.
      • He W.
      • Zhou H.
      • Wang X.
      • Pan D.W.
      • Schimmel P.
      • Pan T.
      • Yang X.-L.
      Evolutionary gain of alanine mischarging to noncognate tRNAs with a G4:U69 base pair.
      ). An Animalia-specific tRNA deacylase was recently discovered that co-occurs with tRNAThr G4:U69 variants in animal genomes and de-acylates mis-charged Ala-tRNAThr (
      • Kuncha S.K.
      • Mazeed M.
      • Singh R.
      • Kattula B.
      • Routh S.B.
      • Sankaranarayanan R.
      A chiral selectivity relaxed paralog of DTD for proofreading tRNA mischarging in Animalia.
      ). This enzyme may protect human cells from alanine mistranslation at threonine codons.
      In our work on tRNA-dependent mistranslation, we expressed a mutant of human tRNAPro containing a G3:U70 base pair in human cells. The human tRNAPro mutant was an efficient alanine acceptor in vitro that no longer accepted proline. Our previous work in yeast demonstrated that a homologous tRNAPro mutant mistranslated multiple proline codons with alanine (
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ). We developed a GFP reporter (D129P) that fluoresces in response to mistranslation at the Pro-129 codon. In HEK 293 cells (
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      ), we did not observe significant mistranslation in rich media compared with cells expressing WT tRNAPro. When the cells were starved of serum and glucose over a period of days, mistranslation accumulated to 2–5% according to the GFP reporter. Strikingly, and similar to the report of Santos et al. (
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      ), we did not observe a loss in cellular viability or induction of the heat-shock response compared with cells expressing the WT tRNA, indicating that this level of mistranslation was well tolerated. In yeast, a mistranslating tRNA did show synthetic slow growth when coupled with deletion mutants lacking the proteome regulatory transcription factor Rpn4 (
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ). The above examples illustrate how phenotypes driven by a mistranslating tRNA variant can remain hidden; yet, in conditions of stress or in models of disease, an otherwise neutral tRNA variant can interact synthetically with a stressor or a second mutation to cause phenotypic defects.
      Errors in protein synthesis are normally thought to be deleterious, yet mistranslation is an adaptive response in diverse organisms from bacteria (
      • Pezo V.
      • Metzgar D.
      • Hendrickson T.L.
      • Waas W.F.
      • Hazebrouck S.
      • Döring V.
      • Marlière P.
      • Schimmel P.
      • De Crécy-Lagard V.
      Artificially ambiguous genetic code confers growth yield advantage.
      ) to yeast (
      • Hoffman K.S.
      • Berg M.D.
      • Shilton B.H.
      • Brandl C.J.
      • O'Donoghue P.
      Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
      ) to human cells (
      • Netzer N.
      • Goodenbour J.M.
      • David A.
      • Dittmar K.A.
      • Jones R.B.
      • Schneider J.R.
      • Boone D.
      • Eves E.M.
      • Rosner M.R.
      • Gibbs J.S.
      • Embry A.
      • Dolan B.
      • Das S.
      • Hickman H.D.
      • Berglund P.
      • et al.
      Innate immune and chemically triggered oxidative stress modifies translational fidelity.
      ). In some of these cases mistranslation provides a selective advantage. As reviewed elsewhere (
      • Moghal A.
      • Mohler K.
      • Ibba M.
      Mistranslation of the genetic code.
      ), a recurrent finding is that stress conditions reduce aminoacylation fidelity (
      • Wu J.
      • Fan Y.
      • Ling J.
      Mechanism of oxidant-induced mistranslation by threonyl-tRNA synthetase.
      ). For example, upon oxidative stress, methionyl-tRNA synthetase increases its mis-aminoacylation of noncognate tRNAs up to 10-fold in bacteria and mammalian cells (
      • Netzer N.
      • Goodenbour J.M.
      • David A.
      • Dittmar K.A.
      • Jones R.B.
      • Schneider J.R.
      • Boone D.
      • Eves E.M.
      • Rosner M.R.
      • Gibbs J.S.
      • Embry A.
      • Dolan B.
      • Das S.
      • Hickman H.D.
      • Berglund P.
      • et al.
      Innate immune and chemically triggered oxidative stress modifies translational fidelity.
      ,
      • Lee J.Y.
      • Kim D.G.
      • Kim B.G.
      • Yang W.S.
      • Hong J.
      • Kang T.
      • Oh Y.S.
      • Kim K.R.
      • Han B.W.
      • Hwang B.J.
      • Kang B.S.
      • Kang M.S.
      • Kim M.H.
      • Kwon N.H.
      • Kim S.
      Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.
      ,
      • Gomes A.C.
      • Kordala A.J.
      • Strack R.
      • Wang X.
      • Geslain R.
      • Delaney K.
      • Clark W.C.
      • Keenan R.
      • Pan T.
      A dual fluorescent reporter for the investigation of methionine mistranslation in live cells.
      ); the additional methionine in proteins is thought to protect the proteome from oxidative damage.

      tRNA variation in humans

      Human tRNA variants that likely mistranslate can be readily identified in publicly available sequence data (Table 1, Table 2, S1) (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
      ). Some of these natural variants (
      • Sun L.
      • Gomes A.C.
      • He W.
      • Zhou H.
      • Wang X.
      • Pan D.W.
      • Schimmel P.
      • Pan T.
      • Yang X.-L.
      Evolutionary gain of alanine mischarging to noncognate tRNAs with a G4:U69 base pair.
      ) or similar variants designed in the laboratory (
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      ,
      • Santos M.
      • Pereira P.M.
      • Varanda A.S.
      • Carvalho J.
      • Azevedo M.
      • Mateus D.D.
      • Mendes N.
      • Oliveira P.
      • Trindade F.
      • Pinto M.T.
      • Bordeira-Carriço R.
      • Carneiro F.
      • Vitorino R.
      • Oliveira C.
      • Santos M.A.S.
      Codon misreading tRNAs promote tumor growth in mice.
      ) do in fact cause mistranslation in human cells. In addition, tRNA variants, which simply impair tRNA folding or function, also impact the proteome. The absence of even a single tRNA gene product can alter the tRNA pool and limit the rate of protein synthesis by causing ribosome stalling (
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ). In both capacities, mutations in tRNA-encoding genes represent an important class of potential disease modifiers that could increase the severity of other disease-causing alleles.
      To visualize the nature and extent of human tRNA variation, we analyzed data from the 1000 Genomes Project and plotted the number of unique variants at each position in an alignment of all human tRNA genes (Fig. 3, A and B). The number of unique variants were mapped on the tRNA secondary structure (Fig. 3A). We also plotted the frequency of occurrence of each of these tRNA mutations in the human population (Fig. 3C). Some tRNAs, such as those for leucine, serine, and selenocysteine, have significantly larger variable loops; variation from these regions was not included.
      Figure thumbnail gr3
      Figure 3tRNA variants observed in the 1000 Genomes Project. A, variants that occur within tRNA genes (defined by GtRNAdb (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      )) were downloaded from the 1000 Genomes Project phase 3 dataset (
      • Auton A.
      • Brooks L.D.
      • Durbin R.M.
      • Garrison E.P.
      • Kang H.M.
      • Korbel J.O.
      • Marchini J.L.
      • McCarthy S.
      • McVean G.A.
      • Abecasis G.R.
      1000 Genomes Project Consortium
      A global reference for human genetic variation.
      ). Insertions and deletions were removed, as were variants with no allele frequency available. Each variant was mapped to its corresponding tRNA position, according to standardized numbering (
      • Sprinzl M.
      • Vassilenko K.S.
      Compilation of tRNA sequences and sequences of tRNA genes.
      ), using an in-house Perl script. High-confidence tRNAs were defined as tRNAs with a tRNAscan-SE score of >50 (
      • Chan P.P.
      • Lowe T.M.
      GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.
      ). For the high confidence tRNA set, unique mutations are mapped to each position in the tRNA. B, same data in A are plotted for the high-confidence set (cyan dashed line) and for all human tRNA sequences (red line). C, allele frequencies (log2 scale) of all variants that occur at each tRNA position are represented in box and whisker plots. Boxes outline quartiles of the allele frequency distribution; filled circles depict the median allele frequency; whiskers show 1.5× quartile range; and open circles depict raw data, i.e. the allele frequencies for each unique tRNA variant at the indicated position.
      No position in human tRNA genes is immune to variation, yet some positions are far more variable than others (Fig. 3, A and B). The allele frequency of these variants indicates that common (>5% allele frequency) and rare variants (<5% allele frequency) are distributed across nearly all sites in the tRNA (Fig. 3C). Some sites, however, lack common variants. Although restricted to rare variants, variation is observed at position 73; this “discriminator” base is a key identity element for many AARSs. The anticodon shows variation at all three bases, albeit reduced compared with other regions of the tRNA. The data from 1000 Genomes Project suggest that across all ∼600 tRNA loci there are 25–30 unique nucleotide variants at each anticodon base, and these include both common and rare variants in the population (Fig. 3C).
      Positions within the acceptor stem contain large numbers of unique variants. Many AARSs recognize acceptor stem nucleotides to ensure aminoacylation fidelity (
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ); thus, acceptor stem variants have the potential to elicit mistranslation or lead to a defective tRNA. Another compelling observation is that several important sites of tRNA modification display significant variation (e.g. position 37 in the anticodon loop) (Fig. 3). Consistent with this observation, mutations in tRNA-modifying enzymes that act at these positions are implicated in disease (
      • Torres A.G.
      • Batlle E.
      • Ribas de Pouplana L.
      Role of tRNA modifications in human diseases.
      ).
      As mentioned above, most human AARSs recognize identity determinants in the tRNA anticodon except for AlaRS, LeuRS, and SerRS (
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ); thus, nonsynonymous anticodon variants in Ala, Leu, and Ser tRNAs are likely to mistranslate. Correspondingly, the tRNA variants that have already been shown to elicit mistranslation in human cells have mutations in the tRNA anticodon or create the identity determinants for AlaRS (
      • Lant J.T.
      • Berg M.D.
      • Sze D.H.W.
      • Hoffman K.S.
      • Akinpelu I.C.
      • Turk M.A.
      • Heinemann I.U.
      • Duennwald M.L.
      • Brandl C.J.
      • O'Donoghue P.
      Visualizing tRNA-dependent mistranslation in human cells.
      ,
      • Hou Y.-M.
      • Francklyn C.
      • Schimmel P.
      Molecular dissection of a transfer RNA and the basis for its identity.
      ,
      • Sun L.
      • Gomes A.C.
      • He W.
      • Zhou H.
      • Wang X.
      • Pan D.W.
      • Schimmel P.
      • Pan T.
      • Yang X.-L.
      Evolutionary gain of alanine mischarging to noncognate tRNAs with a G4:U69 base pair.
      ). Anticodon mutations in other tRNAs typically reduce or ablate aminoacylation (
      • Giegé R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ). However, the degree of aminoacylation loss is not known for all anticodon positions in all tRNAs, and in some cases, efficient aminoacylation can be retained even when some identity determinants are mutated (
      • Fechter P.
      • Rudinger-Thirion J.
      • Tukalo M.
      • Giegé R.
      Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNA(Tyr) are conserved but expressed differently.
      ).
      Certainly, mistranslating tRNAs can arise from a variety of mechanisms. Mutations at identity elements, which ensure cognate aminoacylation, or mutations at anti-determinants, which prevent non-cognate aminoacylation, have the potential to convert any tRNA into a mistranslator. Although such mutants likely occur in the human population, there is a paucity of biochemical data regarding human tRNA identity elements and anti-determinants, thus challenging confident identification of such variants as mistranslating tRNAs from sequence alone. Anticodon mutations in Ser, Ala, and Leu, however, will undoubtedly lead to amino acid mis-incorporation. For this reason, our discussion of specific mistranslating tRNA examples from human genomes focused on these “obvious” mistranslating tRNAs.
      To assess the prevalence of likely tRNA mistranslators in the human population, we searched the Genomic tRNA database (GtRNAdb) for high-confidence Ala, Leu, and Ser tRNAs with anticodon mutations (Table 1) or mutations in the 3rd or 4th acceptor stem bp that create the G3:U70 (Table 1) or G4:U69 (Table S1) AlaRS identity element. In total, among the human Ala, Leu, and Ser iso-acceptor groups reported in GtRNAdb there are 27 unique anticodon variants. Of these, there are 14 unique nonsynonymous (Table 1) and 13 synonymous anticodon variants. Most nonsynonymous anticodon variants are rare, but three variants occur in >1% of the population. One alanine tRNA variant containing a glycine anticodon occurs in over 6% of sequenced individuals. The common occurrence of these mutations in cytosolic tRNAs is striking; analogous variants in mitochondrial tRNAs are embryonic lethal (
      • Kirchner S.
      • Ignatova Z.
      Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.
      ). Although we found a similar number of unique synonymous anticodon variants, none were found in >1% of sequenced individuals. While still encoding the “correct” amino acid, such mutants may more or less efficiently read synonymous codons for a particular amino acid, altering translation rates.
      Further complicating this scenario, certain apparently synonymous anticodon variants may become mistranslators through nucleotide modification. For example, tRNAs normally containing an A34 are modified to inosine (I34) by the action of adenine deaminases acting on tRNAs (
      • Torres A.G.
      • Piñeyro D.
      • Rodríguez-Escribà M.
      • Camacho N.
      • Reina O.
      • Saint-Léger A.
      • Filonava L.
      • Batlle E.
      • Ribas de Pouplana L.
      Inosine modifications in human tRNAs are incorporated at the precursor tRNA level.
      ). As noted above, I34 enables expanded wobble decoding to codons ending in U, C, or A; thus, A34 containing tRNAs are normally restricted to those amino acids with synonymous codons ending in U, C, and A. However, human genomes include examples of tRNAs bearing A34 that, if modified to I34, would lead to mistranslation (e.g. tRNA-Ser-GCT-5–1 single nucleotide polymorphism (SNP) rs550301646; tRNAAsn-ATT-1–1 and tRNA-Tyr-ATA-1–1 are in the human reference genome). In the case of tRNAAsn, A34I would incorporate Asn at Lys AAA codons. This type of phenomenon was recently examined with anticodon variants of Methanocaldococcus jannaschii tRNATyr expressed in E. coli (
      • Biddle W.
      • Schmitt M.A.
      • Fisk J.D.
      Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.
      ,
      • Biddle W.
      • Schmitt M.A.
      • Fisk J.D.
      Evaluating sense codon reassignment with a simple fluorescence screen.
      ). In this case, a tRNATyr mutant with an AUG anticodon decoded histidine CAU and CAC codons with tyrosine at approximately equal efficiency (2–3%); the mutant tRNATyr AUG anticodon was indeed partially modified to IUG (
      • Biddle W.
      • Schmitt M.A.
      • Fisk J.D.
      Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.
      ). Perhaps as a natural defense against mistranslation and the resulting abundant Gln-tRNAUUGGln1, mis-incorporation of tyrosine at glutamine CAA codons was not detected in E. coli (
      • Biddle W.
      • Schmitt M.A.
      • Fisk J.D.
      Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.
      ).
      Adding to the complexity of human tRNA variation, tRNA genes are particularly susceptible to transcription-associated mutagenesis (TAM) (
      • Thornlow B.P.
      • Hough J.
      • Roger J.M.
      • Gong H.
      • Lowe T.M.
      • Corbett-Detig R.B.
      Transfer RNA genes experience exceptionally elevated mutation rates.
      ), and thus, their sequence can change more rapidly than other genes. Thornlow et al. (
      • Thornlow B.P.
      • Hough J.
      • Roger J.M.
      • Gong H.
      • Lowe T.M.
      • Corbett-Detig R.B.
      Transfer RNA genes experience exceptionally elevated mutation rates.
      ) demonstrated that tRNA genes experience 7–10-fold higher rates of TAM compared with the genome-wide average. TAM occurs when DNA strands are separated during transcription, and the nontemplate strand becomes temporarily isolated and more accessible to mutagens (
      • Jinks-Robertson S.
      • Bhagwat A.S.
      Transcription-associated mutagenesis.
      ). Although tRNA variation is generally selected against on a population scale, this implies that the sequence of tRNA genes within individual cells could change throughout life and that perturbations that increase tRNA expression could further increase mutation rates.

      tRNA variation and disease

      Like the involvement of mitochondrial tRNA variants in disease (
      • Abbott J.A.
      • Francklyn C.S.
      • Robey-Bond S.M.
      Transfer RNA and human disease.
      ), recent studies have identified specific cytosolic tRNA mutants as drivers or modifiers of disease in humans and mice. In addition, tRNA mis-modification and imbalanced tRNA expression also contribute to disease. Here, we highlight examples of defective tRNA function in genetic disorders, cancers, and neurodegeneration.

      tRNA mutants linked to disease

      Kobayashi et al. (
      • Kobayashi Y.
      • Momoi M.Y.
      • Tominaga K.
      • Momoi T.
      • Nihei K.
      • Yanagisawa M.
      • Kagawa Y.
      • Ohta S.
      A point mutation in the mitochondrial tRNA(Leu)(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes).
      ) identified the first human tRNA associated with disease in 1990. The mutation, a variant of a mitochondrial tRNALeu gene, leads to the degradation of the tRNA and causes a rare disorder characterized by stroke and dementia: mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. Shortly after, a mutation in mitochondrial tRNALys was found to cause another rare neurological disorder, myoclonic epilepsy and ragged-red fiber disease (
      • Shoffner J.M.
      • Lott M.T.
      • Lezza A.M.
      • Seibel P.
      • Ballinger S.W.
      • Wallace D.C.
      Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation.
      ). Several other mitochondrial tRNA variants are implicated in major human diseases, including heart disease (
      • Jia Z.
      • Wang X.
      • Qin Y.
      • Xue L.
      • Jiang P.
      • Meng Y.
      • Shi S.
      • Wang Y.
      • Qin Mo J.
      • Guan M.-X.
      Coronary heart disease is associated with a mutation in mitochondrial tRNA.
      ), hypertension (
      • Li H.
      • Geng J.
      • Yu H.
      • Tang X.
      • Yang X.
      • Xue L.
      Mitochondrial tRNAThr 15909A>G mutation associated with hypertension in a Chinese Han pedigree.
      ), metabolic disease (
      • Ding Y.
      • Xia B.-H.
      • Zhang C.-J.
      • Zhuo G.-C.
      Mitochondrial tRNALeu(UUR) C3275T, tRNAGln T4363C and tRNALys A8343G mutations may be associated with PCOS and metabolic syndrome.
      ), and deafness (
      • Meng F.
      • He Z.
      • Tang X.
      • Zheng J.
      • Jin X.
      • Zhu Y.
      • Ren X.
      • Zhou M.
      • Wang M.
      • Gong S.
      • Mo J.Q.
      • Shu Q.
      • Guan M.-X.
      Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation.
      ).
      Two clear examples have emerged where a cytosolic tRNA variant either contributes to or directly causes disease. One case (described under “tRNA variants in neurodegeneration”) involves a mutation in a single tRNAArg gene that causes widespread neurodegeneration in mice when associated with a second mutation in a protein-coding gene that sensitizes cells to ribosome stalling (
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ). The other case (detailed under “tRNA modification defects in disease”) is a single nucleotide mutation in the only functional human tRNASec gene (
      • O'Neill V.A.
      • Eden F.C.
      • Pratt K.
      • Hatfield D.L.
      A human opal suppressor tRNA gene and pseudogene.
      ,
      • Santesmasses D.
      • Mariotti M.
      • Guigó R.
      Computational identification of the selenocysteine tRNA (tRNASec) in genomes.
      ) that causes abdominal pain, fatigue, muscle weakness, and low plasma selenium levels in a homozygous patient (
      • Schoenmakers E.
      • Carlson B.
      • Agostini M.
      • Moran C.
      • Rajanayagam O.
      • Bochukova E.
      • Tobe R.
      • Peat R.
      • Gevers E.
      • Muntoni F.
      • Guicheney P.
      • Schoenmakers N.
      • Farooqi S.
      • Lyons G.
      • Hatfield D.
      • Chatterjee K.
      Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.
      ). In this case, the tRNASec variant appears to be the primary driver of disease (
      • Schoenmakers E.
      • Carlson B.
      • Agostini M.
      • Moran C.
      • Rajanayagam O.
      • Bochukova E.
      • Tobe R.
      • Peat R.
      • Gevers E.
      • Muntoni F.
      • Guicheney P.
      • Schoenmakers N.
      • Farooqi S.
      • Lyons G.
      • Hatfield D.
      • Chatterjee K.
      Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.
      ).

      Imbalanced tRNA expression and disease

      tRNA copy number variation

      The copy number of tRNA genes varies between individuals. Iben and Maraia (
      • Iben J.R.
      • Maraia R.J.
      tRNA gene copy number variation in humans.
      ) assessed copy number variation among nuclearly encoded tRNAs from whole-genome sequencing data obtained in the 1000 Genomes Project. Their study focused on two sets of two parents and a child, from which >15-fold read coverage was obtained. Although the high similarity of tRNA iso-acceptors complicates this type of analysis, significant copy number variation in at least 11 tRNA gene loci among the six individuals was reported. Furthermore, they validated a homozygous deletion encoding tRNAUUULys on chromosome 7 in one individual. Interestingly, modification defects in this tRNA associate with type 2 diabetes in mice. The deletion is common in the human population and has no known indication of an associated phenotypic defect (
      • Wei F.-Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ).

      Tissue-specific tRNA expression

      Dittmar et al. (
      • Dittmar K.A.
      • Goodenbour J.M.
      • Pan T.
      Tissue-specific differences in human transfer RNA expression.
      ) demonstrated the tissue dependence of tRNA expression using a tRNA microarray that probed 42 nuclearly encoded and 21 mitochondrially encoded tRNAs from eight different tissues. They revealed that human tissues express different sets of cytosolic tRNAs. Comprehensive analysis based on RNA polymerase III occupancy of tRNA genes in mice support this finding (
      • Kutter C.
      • Brown G.D.
      • Gonçalves A.
      • Wilson M.D.
      • Watt S.
      • Brazma A.
      • White R.J.
      • Odom D.T.
      Pol III binding in six mammals shows conservation among amino acid isotypes despite divergence among tRNA genes.
      ).
      The relevance of tissue-specific tRNA expression to disease was demonstrated by the link between tRNA abundance and cystic fibrosis (
      • Kirchner S.
      • Cai Z.
      • Rauscher R.
      • Kastelic N.
      • Anding M.
      • Czech A.
      • Kleizen B.
      • Ostedgaard L.S.
      • Braakman I.
      • Sheppard D.N.
      • Ignatova Z.
      Alteration of protein function by a silent polymorphism linked to tRNA abundance.
      ). In this work, Kirchner et al. (
      • Kirchner S.
      • Cai Z.
      • Rauscher R.
      • Kastelic N.
      • Anding M.
      • Czech A.
      • Kleizen B.
      • Ostedgaard L.S.
      • Braakman I.
      • Sheppard D.N.
      • Ignatova Z.
      Alteration of protein function by a silent polymorphism linked to tRNA abundance.
      ) characterized a synonymous SNP in the cystic fibrosis transmembrane conductance regulator (CFTR), which substitutes an ACT Thr codon with ACG. This synonymous mutation results in a cell type–dependent alteration of CFTR protein levels that are not explained by a change in mRNA stability or splicing. The authors discovered that tRNACGUThr is a low abundance tRNA in the cystic fibrosis model and human bronchial epithelial cell lines. The polymorphism not only reduces CFTR expression in human bronchial epithelial cells, but also impairs the folding, localization, and membrane conductance of CFTR. The findings point to a translation rate-dependent mechanism, where ribosome stalling on the ACG codon, which is read by a low abundance tRNA, causes the protein product to mis-fold and malfunction.
      Phenotypic defects from synonymous codon mutations are observed in numerous other disease-relevant protein–coding genes (
      • Rauscher R.
      • Ignatova Z.
      Timing during translation matters: synonymous mutations in human pathologies influence protein folding and function.
      ). Examples include multidrug resistance 1 (MDR1) (
      • Kimchi-Sarfaty C.
      • Oh J.M.
      • Kim I.W.
      • Sauna Z.E.
      • Calcagno A.M.
      • Ambudkar S.V.
      • Gottesman M.M.
      A “Silent” polymorphism in the MDR1 gene changes substrate specificity.
      ), estrogen receptor α (
      • Fernández-Calero T.
      • Cabrera-Cabrera F.
      • Ehrlich R.
      • Marín M.
      Silent polymorphisms: can the tRNA population explain changes in protein properties?.
      ,
      • Horjales S.
      • Cota G.
      • Señorale-Pose M.
      • Rovira C.
      • Román E.
      • Artagaveytia N.
      • Ehrlich R.
      • Marín M.
      Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor α.
      ), and surfactant protein-D (
      • Foreman M.G.
      • Kong X.
      • DeMeo D.L.
      • Pillai S.G.
      • Hersh C.P.
      • Bakke P.
      • Gulsvik A.
      • Lomas D.A.
      • Litonjua A.A.
      • Shapiro S.D.
      • Tal-Singer R.
      • Silverman E.K.
      Polymorphisms in surfactant protein-D are associated with chronic obstructive pulmonary disease.
      ). Thus, expression of specific tRNA iso-decoders is an important consideration when synonymous mutations result in a phenotype, particularly if the protein synthesis burden is shifted to a low abundance or possibly defective tRNA. Conceivably, tRNA synonymous anticodon variants (noted above) could have a similar effect on translation rates and cellular phenotypes.

      De-regulated tRNA expression

      tRNA expression can change dynamically in disease. Pavon-Eternod et al. (
      • Pavon-Eternod M.
      • Gomes S.
      • Rosner M.R.
      • Pan T.
      Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells.
      ) demonstrated that tRNA expression increases from 3- to 10-fold in breast cancer tumors. Oncogenic factors such as RAS and C-MYC promote RNA polymerase (pol) III transcription, whereas tumor suppressors such as retinoblastoma protein (RB) and P53 inhibit pol III transcription, providing a link between common cancer mechanisms and pol III-dependent tRNA expression (
      • Huang S.Q.
      • Sun B.
      • Xiong Z.P.
      • Shu Y.
      • Zhou H.H.
      • Zhang W.
      • Xiong J.
      • Li Q.
      The dysregulation of tRNAs and tRNA derivatives in cancer.
      ). Although cause or effect has not been established in these cases, tRNA expression changes in cancer may occur through global tRNA up-regulation to facilitate increased protein synthesis requirements in tumor cells (
      • Mahlab S.
      • Tuller T.
      • Linial M.
      Conservation of the relative tRNA composition in healthy and cancerous tissues.
      ).
      Dysregulation of specific tRNA iso-acceptors is also implicated in cancer. Overexpression of the initiator tRNAMet promotes translation reprogramming and cell proliferation in the human breast epithelial cell lines 184A1 and MCF10A (
      • Pavon-Eternod M.
      • Gomes S.
      • Rosner M.R.
      • Pan T.
      Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells.
      ). This was corroborated in a comprehensive study that quantified tRNA expression profiles using tRNA microarrays and histone modification mapping across 470 patient-derived tissue samples representing various states of proliferation (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ). Gingold et al. (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ) demonstrated that tRNAiMet expression is highest in the most proliferating samples and lowest in the differentiating cells.
      In contrast, reduced tRNASec expression was observed in many proliferating and especially cancerous cell samples (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ). tRNASec is required for the production of selenocysteine-containing proteins. Depending on the context, selenoprotein synthesis can either prevent or promote cancer (
      • Hatfield D.L.
      • Yoo M.-H.
      • Carlson B.A.
      • Gladyshev V.N.
      Selenoproteins that function in cancer prevention and promotion.
      ); thus, up- or down-regulation of tRNASec may have relevance to disease. tRNA expression changes can also promote cancer through roles for tRNAs beyond protein synthesis. A recent review highlighted examples of tRNAs or tRNA-derived fragments from at least 16 iso-acceptor groups that are specifically de-regulated in cancer (
      • Huang S.Q.
      • Sun B.
      • Xiong Z.P.
      • Shu Y.
      • Zhou H.H.
      • Zhang W.
      • Xiong J.
      • Li Q.
      The dysregulation of tRNAs and tRNA derivatives in cancer.
      ).
      The tRNA expression profile in a particular cell will lead to more or less efficient translation of certain mRNAs depending on codon usage (
      • Quax T.E.
      • Claassens N.J.
      • Söll D.
      • van der Oost J.
      Codon bias as a means to fine-tune gene expression.
      ,
      • Frumkin I.
      • Lajoie M.J.
      • Gregg C.J.
      • Hornung G.
      • Church G.M.
      • Pilpel Y.
      Codon usage of highly expressed genes affects proteome-wide translation efficiency.
      ). Differential expression of tRNAs also promotes cancer through favoring particular “translation programs.” The study of Gingold et al. (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ) profiled codon usage in transcripts associated with cell cycle versus differentiation. The authors observed a dichotomy where codons with A or U in the 3rd codon position are generally more common in proliferation-associated mRNAs, and G- or C-ending codons are more common in differentiation-associated mRNA transcripts (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ). The emerging view is that cells dynamically switch between “programs” of protein synthesis, in part by coordinating the transcription of tRNAs with anticodons matching the codon bias in expressed mRNAs.

      Differential expression of specific tRNA iso-decoders

      The expression of specific tRNA iso-decoders promotes metastasis in breast cancer model cell lines (
      • Gingold H.
      • Tehler D.
      • Christoffersen N.R.
      • Nielsen M.M.
      • Asmar F.
      • Kooistra S.M.
      • Christophersen N.S.
      • Christensen L.L.
      • Borre M.
      • Sørensen K.D.
      • Andersen L.D.
      • Andersen C.L.
      • Hulleman E.
      • Wurdinger T.
      • Ralfkiær E.
      • et al.
      A dual program for translation regulation in cellular proliferation and differentiation.
      ). The authors measured the relative abundance of different tRNA iso-decoders in cell lines selected for high rates of metastasis (MDA-LM2 and CN-LM1) and parental cell lines (MDA-231 and CN34). Two tRNAs (tRNACCGArg and tRNAUUCGlu) were highly up-regulated in both metastatic lines. These tRNAs were then overexpressed in MDA-231 cells to assess changes in the proteome resulting from their increased expression. The abundance of proteins encoded by transcripts enriched in the matching codons (GGC and GAR) increased. As measured by ribosome profiling, two such mRNAs (encoding EXOSC2 and GRIPAP1) showed higher rates of active translation in the cells overexpressing tRNAUUCGlu. RNAi-mediated knockdown of these mRNAs reduced in vitro invasion capacity of the cells, suggesting that EXOSC2 and GRIPAP1 are required for tRNAUUCGlu-promoted metastasis. Hence, the coordinated expression of tRNA iso-decoders facilitates translational reprogramming in cancer cells and is implicated in the promotion of proliferation as well as metastasis.

      tRNA modification defects in disease

      As mentioned previously, post-transcriptional modifications are important for tRNA function and stability. Hypo-modification can lead to rapid tRNA decay (
      • Alexandrov A.
      • Chernyakov I.
      • Gu W.
      • Hiley S.L.
      • Hughes T.R.
      • Grayhack E.J.
      • Phizicky E.M.
      Rapid tRNA decay can result from lack of nonessential modifications.
      ), and many tRNAs require anticodon modifications to ensure faithful codon recognition (
      • Crick F.H.
      Codon–anticodon pairing: the wobble hypothesis.
      ,
      • Towns W.L.
      • Begley T.J.
      Transfer RNA methyltransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health.
      ). Over 50 different nucleotide modifications occur in eukaryotic tRNAs (
      • Boccaletto P.
      • Machnicka M.A.
      • Purta E.
      • Piatkowski P.
      • Baginski B.
      • Wirecki T.K.
      • de Crécy-Lagard V.
      • Ross R.
      • Limbach P.A.
      • Kotter A.
      • Helm M.
      • Bujnicki J.M.
      MODOMICS: a database of RNA modification pathways. 2017 update.
      ), and in humans, tRNAs contain an average of 13 modifications per molecule (
      • Saikia M.
      • Fu Y.
      • Pavon-Eternod M.
      • He C.
      • Pan T.
      Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs.
      ). Accordingly, tRNA modification defects are implicated in numerous diseases, including neurological, cardiac, respiratory, and metabolic diseases, as well as cancer and mitochondria-linked disorders (
      • Torres A.G.
      • Batlle E.
      • Ribas de Pouplana L.
      Role of tRNA modifications in human diseases.
      ). Most diseases that result from defects in tRNA modification are due to mutations in protein-coding genes or in mitochondrial tRNA genes, rather than cytosolic tRNA genes.
      A recent example, however, provides compelling evidence of a cytosolic tRNA mutant and de-regulated nucleotide modification in human disease. A C65G mutation in tRNAUCASec was identified in a patient exhibiting abdominal pain, fatigue, muscle weakness, and low plasma selenium levels (
      • Abbott J.A.
      • Francklyn C.S.
      • Robey-Bond S.M.
      Transfer RNA and human disease.
      ). Although humans encode two tRNASec genes, apparently only one is functional. A mutation in this gene has the potential to impact all 25 human selenoproteins, which are essential for normal development (
      • Schweizer U.
      • Fradejas-Villar N.
      Why 21? The significance of selenoproteins for human health revealed by inborn errors of metabolism.
      ). Selenoproteins may be categorized into two groups: housekeeping and stress-related. Synthesis of housekeeping selenoproteins depends on a 5-methoxycarbonylmethyluridine (mcm5U) modification at position 34 of tRNASec, whereas further modification to 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um) promotes synthesis of stress-related selenoproteins (
      • Carlson B.
      • Yoo M.-H.
      • Tsuji P.
      • Gladyshev V.N.
      • Hatfield D.L.
      Mouse models targeting selenocysteine tRNA expression for elucidating the role of selenoproteins in health and development.
      ). The tRNASec C65G variant only impaired expression of stress-related selenoproteins. This is attributed to the fact that the variant has markedly reduced levels of both the mcm5Um modification at position 34 and the N6-isopentenyl adenosine modification at position 37 (
      • Schoenmakers E.
      • Carlson B.
      • Agostini M.
      • Moran C.
      • Rajanayagam O.
      • Bochukova E.
      • Tobe R.
      • Peat R.
      • Gevers E.
      • Muntoni F.
      • Guicheney P.
      • Schoenmakers N.
      • Farooqi S.
      • Lyons G.
      • Hatfield D.
      • Chatterjee K.
      Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.
      ). The finding underscores the complexity of nucleotide modification in tRNA function by showing that a mutation at one site in tRNA can impact modification at other locations in the tRNA body. In this case, a single nucleotide variant in the T-arm altered modifications in the anticodon stem loop (Fig. 1). Although the mechanism is not yet defined, presumably the C65G mutant inhibits or reduces the methyltransferase activity of the multifunction ALKBH8 gene product that catalyzes conversion of mcm5U to mcm5Um at position 34 (
      • Songe-Møller L.
      • van den Born E.
      • Leihne V.
      • Vågbo C.B.
      • Kristoffersen T.
      • Krokan H.E.
      • Kirpekar F.
      • Falnes P.Ø.
      • Klungland A.
      Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.
      ).
      Modifications can also drive or favor specific translation programs (
      • Dedon P.C.
      • Begley T.J.
      A system of RNA modifications and biased codon use controls cellular stress response at the level of translation.
      ,
      • Endres L.
      • Dedon P.C.
      • Begley T.J.
      Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses.
      ). For example, melanomas harboring the V600E mutation in the proto-oncoprotein B-RAF depend on translational reprogramming controlled by up-regulation of U34 tRNA-modifying enzymes (
      • Rapino F.
      • Delaunay S.
      • Rambow F.
      • Zhou Z.
      • Tharun L.
      • De Tullio P.
      • Sin O.
      • Shostak K.
      • Schmitz S.
      • Piepers J.
      • Ghesquière B.
      • Karim L.
      • Charloteaux B.
      • Jamart D.
      • Florin A.
      • et al.
      Codon-specific translation reprogramming promotes resistance to targeted therapy.
      ). Similar to the modulation of tRNA expression in metastatic breast cancer (
      • Goodarzi H.
      • Nguyen H.C.B.
      • Zhang S.
      • Dill B.D.
      • Molina H.
      • Tavazoie S.F.
      Modulated expression of specific tRNAs drives gene expression and cancer progression.
      ), the mechanism relies on coordinated regulation of both tRNAs and associated codon-biased transcripts. These modification tunable transcripts are sensitive to particular tRNA modification states (
      • Dedon P.C.
      • Begley T.J.
      A system of RNA modifications and biased codon use controls cellular stress response at the level of translation.
      ). U34 tRNA modification promotes decoding of the “-AA” ending codons AAA, GAA, and CAA (
      • Nedialkova D.D.
      • Leidel S.A.
      Optimization of codon translation rates via tRNA modifications maintains proteome integrity.
      ). Remarkably, up-regulation of U34-modifying enzymes promotes survival of melanomas dependent on hypoxia inducible factor 1α (HIF-1α) metabolism. Elevated levels of HIF-1α correlate with tumor metastasis and poor patient prognosis as well as tumor resistance to therapy (
      • Masoud G.N.
      • Li W.
      HIF-1α pathway: role, regulation and intervention for cancer therapy.
      ). Indeed, the HIF-1α mRNA is enriched in AAA, GAA, and CAA codons (
      • Rapino F.
      • Delaunay S.
      • Rambow F.
      • Zhou Z.
      • Tharun L.
      • De Tullio P.
      • Sin O.
      • Shostak K.
      • Schmitz S.
      • Piepers J.
      • Ghesquière B.
      • Karim L.
      • Charloteaux B.
      • Jamart D.
      • Florin A.
      • et al.
      Codon-specific translation reprogramming promotes resistance to targeted therapy.
      ). When U34-modifying enzymes ELP3, CTU1, or CTU2 were knocked down, HIF-1α protein levels decreased even though HIF-1α mRNA levels were unchanged. Thus, cancer cells are able to regulate tRNA modification enzymes to ultimately tune protein synthesis rates and protein levels in favor of oncogenesis.

      tRNA variants in neurodegeneration

      A common attribute of disorders linked to defective protein homeostasis is the accumulation of mistranslated or misfolded proteins in cells (
      • Kapur M.
      • Ackerman S.L.
      mRNA translation gone awry: translation fidelity and neurological disease.
      ,
      • Josefson R.
      • Andersson R.
      • Nyström T.
      How and why do toxic conformers of aberrant proteins accumulate during ageing?.
      ). In many cell types, this problem can be counteracted through apoptosis or cell division (
      • Josefson R.
      • Andersson R.
      • Nyström T.
      How and why do toxic conformers of aberrant proteins accumulate during ageing?.
      ). However, post-mitotic cells such as those found in the heart and brain are incapable of diluting misfolded proteins through division and lack the regenerative capacity to replace apoptotic cells readily (
      • Josefson R.
      • Andersson R.
      • Nyström T.
      How and why do toxic conformers of aberrant proteins accumulate during ageing?.
      ). Furthermore, protein quality control decreases in post-mitotic tissues with age (
      • de Toda I.M.
      • Vida C.
      • Ortega E.
      • De La Fuente M.
      Hsp70 basal levels, a tissue marker of the rate of aging and longevity in mice.
      ). Post-mitotic tissues may be particularly vulnerable to the consequences of tRNA variants and increasingly so with age.
      Girstmair et al. (
      • Girstmair H.
      • Saffert P.
      • Rode S.
      • Czech A.
      • Holland G.
      • Bannert N.
      • Ignatova Z.
      Depletion of cognate charged transfer RNA causes translational frameshifting within the expanded CAG stretch in huntingtin.
      ) proposed a role for cytosolic tRNAs in Huntington’s disease (HD). HD is caused by an expanded Gln repeat in the huntingtin protein, encoded by a stretch of 40–100 repeated CAG codons (
      • Bates G.P.
      • Dorsey R.
      • Gusella J.F.
      • Hayden M.R.
      • Kay C.
      • Leavitt B.R.
      • Nance M.
      • Ross C.A.
      • Scahill R.I.
      • Wetzel R.
      • Wild E.J.
      • Tabrizi S.J.
      Huntington disease.
      ). In some cases, shorter CAG repeats appear to also cause HD, suggesting there are additional disease modifiers. Continuous translation of the repeat depletes charged tRNACUGGln, which results in more frequent frameshifting in the translation of the huntingtin gene, possibly exacerbating the disease phenotype (
      • Girstmair H.
      • Saffert P.
      • Rode S.
      • Czech A.
      • Holland G.
      • Bannert N.
      • Ignatova Z.
      Depletion of cognate charged transfer RNA causes translational frameshifting within the expanded CAG stretch in huntingtin.
      ). Although tRNACUGGln variants are not yet known to exacerbate HD, these findings illustrate the importance of tRNA function and abundance in pathologies of the brain.
      Indeed, naturally occurring tRNA variants have the potential to deplete the abundance of a brain-specific tRNA that is essential for health. Ishimura et al. (
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ) uncovered a synthetic toxic effect involving a single cytosolic tRNA variant that causes widespread neurodegeneration in mice (
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      ). Mutations in Gtpbp2 (encoding a protein that rescues stalled ribosomes) and Tr20 (encoding tRNAUCUArg) were found to co-occur in mice identified in a phenotypic screen for neurodegeneration. Mice carrying both mutations exhibit rapid neurodegeneration and die at 8–9 weeks. At 3 weeks, the mutant mice are indistinguishable from WT. The C50T mutation (n-Tr20) prevents tRNAUCUArg maturation and, in combination with the loss of GTPBP2, leads to ribosome stalling. Despite many “redundant” tRNAArg iso-decoders in the cell, the lack of function of this single tRNA causes ribosome stalling. The authors measured a 3-fold increase in AGA pauses in the n-Tr20 mutant compared with a mouse containing the WT tRNA. Fascinatingly, tRNAArg C50T variants also occur in the human population, including in a TCT iso-acceptor (Table 3).
      This work exemplifies the ways in which tRNA variants can exacerbate pathways to disease. Two observations from this work may have broader applicability to understanding the roles for tRNA variants in disease. First, the phenotype was tissue-specific, because expression of the n-Tr20–encoded tRNA is only observed in the central nervous system. Second, a coincident mutation in another gene sensitized cells to the loss-of-function mutation in a single tRNA gene. Together, these mutations caused disease in the animal model.

      Conclusion

      Humans display a remarkable array of both common and rare tRNA mutants, some with the obvious potential to mistranslate the genetic code (Table 1, Table 2 and Table S1) or create defective tRNAs (e.g. Table 3). Indeed, such tRNA variants can elicit significant levels of mistranslation in human cells and influence protein synthesis and protein homeostasis (Fig. 4). Above, we highlighted recent examples showing how tRNA variants and defective tRNA genes contribute to disease. In addition to causing disease, tRNA variants act synergistically with other disease-causing alleles by placing additional stress on protein quality control mechanisms or biasing translation programs that drive disease. Furthermore, tissue-specific tRNA expression and de-regulated tRNA expression or modification contribute to disease and phenotypic defects at the cellular level. Together, these observations suggest that cytosolic tRNA mutations may have greater importance in disease than previously recognized. We hope that the evidence provided in this review will stimulate new interest in considering cytoplasmic tRNA variants as an important factor in human genetic variation and disease.
      Figure thumbnail gr4
      Figure 4Phenotypic consequences of tRNA variation. tRNA variation can impact expression, aminoacylation, or processing and maturation of tRNAs, including nucleotide modification and tRNA folding. Alterations in the functional tRNA pool can impact mRNA translation by causing mistranslation, frame-shifting, ribosome stalling, or increased expression of codon-biased transcripts. These alterations can in turn alter the amino acid sequence, folding, and abundance of proteins across the entire proteome.

      Acknowledgments

      We are grateful to David Wright and Lluís Ribas de Pouplana for insightful discussions and suggestions to the manuscript.

      References

        • Crick F.H.C.
        On degenerate templates and the adaptor hypothesis.
        A Note for the RNA Tie Club. 1955; : 1-17
        • Hoagland M.B.
        • Stephenson M.L.
        • Scott J.F.
        • Hecht L.I.
        • Zamecnik P.C.
        A soluble ribonucleic acid intermediate in protein synthesis.
        J. Biol. Chem. 1958; 231 (13538965): 241-257
        • Holley R.W.
        • Everett G.A.
        • Madison J.T.
        • Zamir A.
        Nucleotide sequences in the yeast alanine transfer ribonucleic acid.
        J. Biol. Chem. 1965; 240 (14299636): 2122-2128
        • Gardner R.S.
        • Wahba A.J.
        • Basilio C.
        • Miller R.S.
        • Lengyel P.
        • Speyer J.F.
        Synthetic polynucleotides and the amino acid code. VII.
        Proc. Natl. Acad. Sci. U.S.A. 1962; 48 (13946552): 2087-2094
        • Nishimura S.
        • Jones D.S.
        • Khorana H.G.
        Studies on polynucleotides. 48. The in vitro synthesis of a co-polypeptide containing two amino acids in alternating sequence dependent upon a DNA-like polymer containing two nucleotides in alternating sequence.
        J. Mol. Biol. 1965; 13 (5323614): 302-324
        • Nirenberg M.
        • Leder P.
        • Bernfield M.
        • Brimacombe R.
        • Trupin J.
        • Rottman F.
        • O'Neal C.
        RNA codewords and protein synthesis, VII. On the general nature of the RNA code.
        Proc. Natl. Acad. Sci. U.S.A. 1965; 53 (5330357): 1161-1168
        • Söll D.
        • Ohtsuka E.
        • Jones D.S.
        • Lohrmann R.
        • Hayatsu H.
        • Nishimura S.
        • Khorana H.G.
        Studies on polynucleotides, XLIX. Stimulation of the binding of aminoacyl-sRNA’s to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids.
        Proc. Natl. Acad. Sci. U.S.A. 1965; 54 (5325653): 1378-1385
        • Carbon J.
        • Berg P.
        • Yanofsky C.
        Missense suppression due to a genetically altered tRNA.
        Cold Spring Harb. Symp. Quant. Biol. 1966; 31 (4866396): 487-497
        • Riyasaty S.
        • Atkins J.F.
        External suppression of a frameshift mutant in Salmonella.
        J. Mol. Biol. 1968; 34 (4938557): 541-557
        • Ling J.
        • O’Donoghue P.
        • Söll D.
        Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology.
        Nat. Rev. Microbiol. 2015; 13 (26411296): 707-721
        • Bezerra A.R.
        • Simões J.
        • Lee W.
        • Rung J.
        • Weil T.
        • Gut I.G.
        • Gut M.
        • Bayés M.
        • Rizzetto L.
        • Cavalieri D.
        • Giovannini G.
        • Bozza S.
        • Romani L.
        • Kapushesky M.
        • Moura G.R.
        • Santos M.A.
        Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23776239): 11079-11084
        • Lant J.T.
        • Berg M.D.
        • Sze D.H.W.
        • Hoffman K.S.
        • Akinpelu I.C.
        • Turk M.A.
        • Heinemann I.U.
        • Duennwald M.L.
        • Brandl C.J.
        • O'Donoghue P.
        Visualizing tRNA-dependent mistranslation in human cells.
        RNA Biol. 2018; 15 (28933646): 567-575
        • Hoffman K.S.
        • Berg M.D.
        • Shilton B.H.
        • Brandl C.J.
        • O'Donoghue P.
        Genetic selection for mistranslation rescues a defective co-chaperone in yeast.
        Nucleic Acids Res. 2017; 45 (27899648): 3407-3421
        • Barrell B.G.
        • Bankier A.T.
        • Drouin J.
        A different genetic code in human mitochondria.
        Nature. 1979; 282 (226894): 189-194
        • Campbell J.H.
        • O’Donoghue P.
        • Campbell A.G.
        • Schwientek P.
        • Sczyrba A.
        • Woyke T.
        • Söll D.
        • Podar M.
        UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23509275): 5540-5545
        • Böck A.
        • Stadtman T.C.
        Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism.
        Biofactors. 1988; 1 (2978458): 245-250
        • Berry M.J.
        • Martin 3rd., G.W.
        • Tujebajeva R.
        • Grundner-Culemann E.
        • Mansell J.B.
        • Morozova N.
        • Harney J.W.
        Selenocysteine insertion sequence element characterization and selenoprotein expression.
        Methods Enzymol. 2002; 347 (11898404): 17-24
        • Bröcker M.J.
        • Ho J.M.
        • Church G.M.
        • Söll D.
        • O’Donoghue P.
        Recoding the genetic code with selenocysteine.
        Angew. Chem. Int. Ed. Engl. 2014; 53 (24511637): 319-323
        • Schoenmakers E.
        • Carlson B.
        • Agostini M.
        • Moran C.
        • Rajanayagam O.
        • Bochukova E.
        • Tobe R.
        • Peat R.
        • Gevers E.
        • Muntoni F.
        • Guicheney P.
        • Schoenmakers N.
        • Farooqi S.
        • Lyons G.
        • Hatfield D.
        • Chatterjee K.
        Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.
        J. Clin. Invest. 2016; 126 (26854926): 992-996
        • Kapur M.
        • Ackerman S.L.
        mRNA translation gone awry: translation fidelity and neurological disease.
        Trends Genet. 2018; 34 (29352613): 218-231
        • Ishimura R.
        • Nagy G.
        • Dotu I.
        • Zhou H.
        • Yang X.L.
        • Schimmel P.
        • Senju S.
        • Nishimura Y.
        • Chuang J.H.
        • Ackerman S.L.
        Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
        Science. 2014; 345 (25061210): 455-459
        • Santos M.
        • Pereira P.M.
        • Varanda A.S.
        • Carvalho J.
        • Azevedo M.
        • Mateus D.D.
        • Mendes N.
        • Oliveira P.
        • Trindade F.
        • Pinto M.T.
        • Bordeira-Carriço R.
        • Carneiro F.
        • Vitorino R.
        • Oliveira C.
        • Santos M.A.S.
        Codon misreading tRNAs promote tumor growth in mice.
        RNA Biol. 2018; 15 (29558247): 773-786
        • Berg M.D.
        • Hoffman K.S.
        • Genereaux J.
        • Mian S.
        • Trussler R.S.
        • Haniford D.B.
        • O'Donoghue P.
        • Brandl C.J.
        Evolving mistranslating tRNAs through a phenotypically ambivalent intermediate in Saccharomyces cerevisiae.
        Genetics. 2017; 206 (28576863): 1865-1879
        • Meyer-Schuman R.
        • Antonellis A.
        Emerging mechanisms of aminoacyl-tRNA synthetase mutations in recessive and dominant human disease.
        Hum. Mol. Genet. 2017; 26 (28633377): R114-R127
        • Bednářová A.
        • Hanna M.
        • Durham I.
        • VanCleave T.
        • England A.
        • Chaudhuri A.
        • Krishnan N.
        Lost in translation: defects in transfer RNA modifications and neurological disorders.
        Front. Mol. Neurosci. 2017; 10 (28536502): 135
        • Torres A.G.
        • Batlle E.
        • Ribas de Pouplana L.
        Role of tRNA modifications in human diseases.
        Trends Mol. Med. 2014; 20 (24581449): 306-314
        • Kirchner S.
        • Ignatova Z.
        Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.
        Nat. Rev. Genet. 2015; 16 (25534324): 98-112
        • Abbott J.A.
        • Francklyn C.S.
        • Robey-Bond S.M.
        Transfer RNA and human disease.
        Front. Genet. 2014; 5 (24917879): 158
        • Yasukawa T.
        • Suzuki T.
        • Ishii N.
        • Ohta S.
        • Watanabe K.
        Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease.
        EMBO J. 2001; 20 (11532943): 4794-4802
        • Agris P.F.
        • Vendeix F.A.
        • Graham W.D.
        tRNA's wobble decoding of the genome: 40 years of modification.
        J. Mol. Biol. 2007; 366 (17187822): 1-13
        • Joshi K.
        • Bhatt M.J.
        • Farabaugh P.J.
        Codon-specific effects of tRNA anticodon loop modifications on translational misreading errors in the yeast Saccharomyces cerevisiae.
        Nucleic Acids Res. 2018; 46 (30060218): 10331-10339
        • Pavlov M.Y.
        • Ehrenberg M.
        Substrate-induced formation of ribosomal decoding center for accurate and rapid genetic code translation.
        Annu. Rev. Biophys. 2018; 47 (29792818): 525-548
        • O’Donoghue P.
        • Prat L.
        • Heinemann I.U.
        • Ling J.
        • Odoi K.
        • Liu W.R.
        • Söll D.
        Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion.
        FEBS Lett. 2012; 586 (23036644): 3931-3937
        • Crick F.H.
        Codon–anticodon pairing: the wobble hypothesis.
        J. Mol. Biol. 1966; 19 (5969078): 548-555
        • Söll D.
        • Jones D.S.
        • Ohtsuka E.
        • Faulkner R.D.
        • Lohrmann R.
        • Hayatsu H.
        • Khorana H.G.
        Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique.
        J. Mol. Biol. 1966; 19 (5338858): 556-573
        • Agris P.F.
        • Narendran A.
        • Sarachan K.
        • Väre V.Y.P.
        • Eruysal E.
        The importance of being modified: the role of RNA modifications in translational fidelity.
        Enzymes. 2017; 41 (28601219): 1-50
        • Gustilo E.M.
        • Vendeix F.A.
        • Agris P.F.
        tRNA's modifications bring order to gene expression.
        Curr. Opin. Microbiol. 2008; 11 (18378185): 134-140
        • Tükenmez H.
        • Xu H.
        • Esberg A.
        • Byström A.S.
        The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes.
        Nucleic Acids Res. 2015; 43 (26283182): 9489-9499
        • Lin C.A.
        • Ellis S.R.
        • True H.L.
        The Sua5 protein is essential for normal translational regulation in yeast.
        Mol. Cell. Biol. 2010; 30 (19884342): 354-363
        • Jaffe J.D.
        • Stange-Thomann N.
        • Smith C.
        • DeCaprio D.
        • Fisher S.
        • Butler J.
        • Calvo S.
        • Elkins T.
        • FitzGerald M.G.
        • Hafez N.
        • Kodira C.D.
        • Major J.
        • Wang S.
        • Wilkinson J.
        • Nicol R.
        • Nusbaum C.
        • et al.
        The complete genome and proteome of Mycoplasma mobile.
        Genome Res. 2004; 14 (15289470): 1447-1461
        • Chatterjee K.
        • Nostramo R.T.
        • Wan Y.
        • Hopper A.K.
        tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: location, location, location.
        Biochim. Biophys. Acta. 2018; 1861: 373-386
        • Goodenbour J.M.
        • Pan T.
        Diversity of tRNA genes in eukaryotes.
        Nucleic Acids Res. 2006; 34 (17088292): 6137-6146
        • Chan P.P.
        • Lowe T.M.
        GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
        Nucleic Acids Res. 2009; 37 (18984615): D93-D97
        • Bour T.
        • Mahmoudi N.
        • Kapps D.
        • Thiberge S.
        • Bargieri D.
        • Ménard R.
        • Frugier M.
        Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites.
        Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27071116): 4717-4722
        • Umu S.U.
        • Langseth H.
        • Bucher-Johannessen C.
        • Fromm B.
        • Keller A.
        • Meese E.
        • Lauritzen M.
        • Leithaug M.
        • Lyle R.
        • Rounge T.B.
        A comprehensive profile of circulating RNAs in human serum.
        RNA Biol. 2018; 15 (29219730): 242-250
        • Canella D.
        • Praz V.
        • Reina J.H.
        • Cousin P.
        • Hernandez N.
        Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells.
        Genome Res. 2010; 20 (20413673): 710-721
        • Gogakos T.
        • Brown M.
        • Garzia A.
        • Meyer C.
        • Hafner M.
        • Tuschl T.
        Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP.
        Cell Rep. 2017; 20 (28793268): 1463-1475
        • Dittmar K.A.
        • Goodenbour J.M.
        • Pan T.
        Tissue-specific differences in human transfer RNA expression.
        PLoS Genet. 2006; 2 (17194224): e221
        • Melé M.
        • Ferreira P.G.
        • Reverter F.
        • DeLuca D.S.
        • Monlong J.
        • Sammeth M.
        • Young T.R.
        • Goldmann J.M.
        • Pervouchine D.D.
        • Sullivan T.J.
        • Johnson R.
        • Segrè A.V.
        • Djebali S.
        • Niarchou A.
        • et al.
        • GTEx Consortium
        Human genomics. The human transcriptome across tissues and individuals.
        Science. 2015; 348 (25954002): 660-665
        • Grote A.
        • Hiller K.
        • Scheer M.
        • Münch R.
        • Nörtemann B.
        • Hempel D.C.
        • Jahn D.
        JCat: a novel tool to adapt codon usage of a target gene to its potential expression host.
        Nucleic Acids Res. 2005; 33 (15980527): W526-W531
        • Betat H.
        • Long Y.
        • Jackman J.E.
        • Mörl M.
        From end to end: tRNA editing at 5′- and 3′-terminal positions.
        Int. J. Mol. Sci. 2014; 15 (25535083): 23975-23998
        • Wilusz J.E.
        Controlling translation via modulation of tRNA levels: controlling translation via modulation of tRNA levels.
        Wiley Interdiscip. Rev. RNA. 2015; 6 (25919480): 453-470
        • Wilusz J.E.
        • Whipple J.M.
        • Phizicky E.M.
        • Sharp P.A.
        tRNAs marked with CCACCA are targeted for degradation.
        Science. 2011; 334 (22076379): 817-821
        • Chakraborty P.K.
        • Schmitz-Abe K.
        • Kennedy E.K.
        • Mamady H.
        • Naas T.
        • Durie D.
        • Campagna D.R.
        • Lau A.
        • Sendamarai A.K.
        • Wiseman D.H.
        • May A.
        • Jolles S.
        • Connor P.
        • Powell C.
        • Heeney M.M.
        • et al.
        Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).
        Blood. 2014; 124 (25193871): 2867-2871
        • Lorenz C.
        • Lunse C.E.
        • Morl M.
        tRNA modifications: impact on structure and thermal adaptation.
        Biomolecules. 2017; 7: 35
        • Heinemann I.U.
        • Nakamura A.
        • O’Donoghue P.
        • Eiler D.
        • Söll D.
        tRNAHis-guanylyltransferase establishes tRNAHis identity.
        Nucleic Acids Res. 2012; 40 (21890903): 333-344
        • Gu W.
        • Hurto R.L.
        • Hopper A.K.
        • Grayhack E.J.
        • Phizicky E.M.
        Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C.
        Mol. Cell. Biol. 2005; 25 (16135808): 8191-8201
        • Preston M.A.
        • D'Silva S.
        • Kon Y.
        • Phizicky E.M.
        tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae.
        RNA. 2013; 19 (23249748): 243-256
        • Martinez A.
        • Yamashita S.
        • Nagaike T.
        • Sakaguchi Y.
        • Suzuki T.
        • Tomita K.
        Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA.
        Nucleic Acids Res. 2017; 45 (28119416): 5423-5436
        • Tomita K.
        • Liu Y.
        Human BCDIN3D is a cytoplasmic tRNA(His)-specific 5′-monophosphate methyltransferase.
        Front. Genet. 2018; 9 (30127802): 305
        • Saikia M.
        • Fu Y.
        • Pavon-Eternod M.
        • He C.
        • Pan T.
        Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs.
        RNA. 2010; 16 (20484468): 1317-1327
        • Schimmel P.
        The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
        Nat. Rev. Mol. Cell Biol. 2018; 19 (28875994): 45-58
        • Towns W.L.
        • Begley T.J.
        Transfer RNA methyltransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health.
        DNA Cell Biol. 2012; 31 (22191691): 434-454
        • Hopper A.K.
        Transfer RNA post-transcriptional processing, turnover, and subcellular dynamics in the yeast Saccharomyces cerevisiae.
        Genetics. 2013; 194 (23633143): 43-67
        • Dedon P.C.
        • Begley T.J.
        A system of RNA modifications and biased codon use controls cellular stress response at the level of translation.
        Chem. Res. Toxicol. 2014; 27 (24422464): 330-337
        • Endres L.
        • Dedon P.C.
        • Begley T.J.
        Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses.
        RNA Biol. 2015; 12 (25892531): 603-614
        • Rapino F.
        • Delaunay S.
        • Rambow F.
        • Zhou Z.
        • Tharun L.
        • De Tullio P.
        • Sin O.
        • Shostak K.
        • Schmitz S.
        • Piepers J.
        • Ghesquière B.
        • Karim L.
        • Charloteaux B.
        • Jamart D.
        • Florin A.
        • et al.
        Codon-specific translation reprogramming promotes resistance to targeted therapy.
        Nature. 2018; 558 (29925953): 605-609
        • Ibba M.
        • Soll D.
        Aminoacyl-tRNA synthesis.
        Annu. Rev. Biochem. 2000; 69 (10966471): 617-650
        • Giegé R.
        • Sissler M.
        • Florentz C.
        Universal rules and idiosyncratic features in tRNA identity.
        Nucleic Acids Res. 1998; 26 (9801296): 5017-5035
        • Payea M.J.
        • Sloma M.F.
        • Kon Y.
        • Young D.L.
        • Guy M.P.
        • Zhang X.
        • De Zoysa T.
        • Fields S.
        • Mathews D.H.
        • Phizicky E.M.
        Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA.
        RNA. 2018; 24 (