GE46CH04-deCrecy ARI 6 August 2012 17:48 R E V I E W S I N A D V A N C E Biosynthesis and Function of Posttranscriptional Modifications of Transfer RNAs Basma El Yacoubi, Marc Bailly, and Val ´ erie de Cr ´ ecy-Lagard Department of Microbiology and Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700; email: vcrecy@ufl.edu Annu. Rev. Genet. 2012. 46:69–95 The Annual Review of Genetics is online at genet.annualreviews.org This article’s doi: 10.1146/annurev-genet-110711-155641 Copyright c 2012 by Annual Reviews. All rights reserved 0066-4197/12/1201-0069$20.00 Keywords tRNA, genetic code, thiolation, ELP, KEOPS, queuosine Abstract Posttranscriptional modifications of transfer RNAs (tRNAs) are crit- ical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to intro- duce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modi- fications and tRNA modification enzymes as checkpoints for tRNA in- tegrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global sur- vey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the so- lutions used to fulfill these constraints differ among different kingdoms, organisms, and species. 69 Review in Advance first posted online on August 16, 2012. (Changes may still occur before final publication online and in print.) Changes may still occur before final publication online and in print Annu. Rev. Genet. 2012.46. Downloaded from www.annualreviews.org by NORTH CAROLINA STATE UNIVERSITY on 09/12/12. For personal use only.
Transcript
GE46CH04-deCrecy ARI 6 August 2012 17:48
RE V I E W
S
IN
AD V A
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Biosynthesis and Functionof PosttranscriptionalModifications ofTransfer RNAsBasma El Yacoubi, Marc Bailly,and Valerie de Crecy-LagardDepartment of Microbiology and Department of Microbiology and Cell Science,University of Florida, Gainesville, Florida 32611-0700; email: [email protected]
Annu. Rev. Genet. 2012. 46:69–95
The Annual Review of Genetics is online atgenet.annualreviews.org
This article’s doi:10.1146/annurev-genet-110711-155641
Posttranscriptional modifications of transfer RNAs (tRNAs) are crit-ical for all core aspects of tRNA function, such as folding, stability,and decoding. Most tRNA modifications were discovered in the 1970s;however, the near-complete description of the genes required to intro-duce the full set of modifications in both yeast and Escherichia coli is veryrecent. This led to a new appreciation of the key roles of tRNA modi-fications and tRNA modification enzymes as checkpoints for tRNA in-tegrity and for integrating translation with other cellular functions suchas transcription, primary metabolism, and stress resistance. A global sur-vey of tRNA modification enzymes shows that the functional constraintsthat drive the presence of modifications are often conserved, but the so-lutions used to fulfill these constraints differ among different kingdoms,organisms, and species.
69
Review in Advance first posted online on August 16, 2012. (Changes may still occur before final publication online and in print.)
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INTRODUCTION
As adaptors between messenger RNAs (mR-NAs) and the elongating peptide chains,transfer RNAs (tRNAs) are central to thedecoding process and interact with most com-ponents of the translation apparatus. Whereaspolypeptides use a chemically diverse set ofbuilding blocks (the 20 proteogenic aminoacids), tRNA molecules are limited to the fourribonucleosides incorporated during transcrip-tion. The chemical diversity of tRNA moleculesis, however, greatly augmented by the additionof posttranscriptional modifications (53), oneof the multiple steps in tRNA maturationthat also includes removal of 3′ and 5′ leadersequences, addition of an essential 3′ CCA tail,and removal of introns (95, 117) (Figure 1).
Because tRNAs decipher the genetic ma-terial, proper decoding by these molecules iscrucial to cell survival. A large proportion ofthe cell’s genome is devoted to ensuring thattRNAs can accomplish this task and nucleosidemodifications are an integral part of this pro-cess. Indeed, 1% to 10% of the genes in a givengenome encode enzymes involved in tRNAmodification, more than the total number oftRNA genes per se, which highlights the im-portance of modifications at all levels of tRNAfunction. Approximately 85 different modifica-tions have been identified in tRNA molecules,with the great majority found at positions 34and 37 in the anticodon stem loop (ASL) (53)(Figure 1 and Table 1), and new ones are stillbeing discovered (79). Comprehensive infor-mation on structures and positions of thesemodifications, their biosynthetic pathways, andenzymes can be found in the RNA modificationdatabase (http://rna-mdb.cas.albany.edu/RNAmods/) (22), the Modomics database(http://modomics.genesilico.pl/) (31), andthe tRNAdb database (http://trnadb.bioinf.uni-leipzig.de/) (71). Abbreviations of thetRNA modifications used in this review arelisted in Supplemental Table 1 (followthe Supplemental Material link from theAnnual Reviews home page at http://www.annualreviews.org).
Even though most tRNA modifications wereidentified over 40 years ago, the discovery of thecorresponding enzymes and genes has laggedbehind (51). This made it difficult to evalu-ate the biological roles of these modifications.Fueled by the genomic revolution and technicaladvances, the identification of most tRNA mod-ification enzymes and genes, at least in modelorganisms, has led to increased appreciationof the importance of tRNA modifications forthe integrity of many components of the cellu-lar machinery (Figure 1) and provided insightsinto the evolution of translation in the differentkingdoms of life.
IDENTIFICATION OF ALLTRANSFER RNA MODIFICATIONGENES IN MODEL ORGANISMSFROM THE THREE KINGDOMS
The technical challenges inherent to the directsequencing of purified tRNA molecules foridentifying the positions and chemical natureof modifications found in tRNAs have beena major limitation in the field. Thousandsof sequences of tRNA genes (or tDNAs) aredirectly available from genome sequences, butonly 606 tRNAs from 579 species have beensequenced (71). In addition, a fully (or nearlyfully) sequenced set has been achieved for onlya handful of organisms, including the bacteriaEscherichia coli (41 sequenced/46 total tRNAspecies) and Mycoplasma capricolum (29/29), theeukaryote Saccharomyces cerevisiae (37/42), andthe archaeon Haloferax volcanii (40/46). As aresult, most experimental efforts have focusedon identifying tRNA modification genes inthese specific organisms. After nearly 40 yearsof studies, most of these genes have beenidentified and experimentally validated in bothS. cerevisiae and E. coli (Figure 2 and Table 1).Only a few genes involved in the formationof complex modifications of the ASL remainmissing in S. cerevisiae (see Table 1). In E.coli, the remaining missing tRNA modificationgenes are the m2A37 methylase gene as wellas the gene(s) for acp3U47 synthesis and forthe formation of the ho5U34 intermediate in
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Figure 1Pivotal roles of nucleoside modification for tRNA function from birth to death. Nucleoside modifications can have structural rolescontributing to the formation of the correct tRNA L shape and are also involved in tRNA interactions with numerous players of thetranslation machinery, such as modification enzymes, mRNA codons, ribosomes, translation factors, AARSs, as well as editing andRNA-degradation systems (TRAMP/exosome, RTD, and endoribonucleases involved in stress responses). Modifications can beintroduced on their tRNA substrates in the nucleus, in the cytoplasm, or in organelles (by modification enzymes encoded in thenucleus). Through this plethora of functions, modifications play a central role in translation accuracy and efficiency, exemplified by themodifications termed essential for life. However, most individual contributions can appear minimal and are revealed only under stressor other specific conditions. Also, many modifications can play redundant roles so that more than one modification needs to be depletedfor a deleterious phenotype to be observed. When translation accuracy and efficiency are impaired owing to lack of tRNAmodifications, the resulting statistical proteome (i.e., the heterogeneous protein pool, with some fraction of proteins containing errors,whereas others are wild type) is thought to be at the origin of the observed phenotypes. Recent studies link tRNA modification andcontrol of gene expression in response to stress; however, the biological basis for this possible regulatory role remains elusive.Abbreviations: AARS, aminoacyl-tRNA synthetase; ASL, anticodon stem loop; DSL, D stem loop; mRNA, messenger RNA; RTD:rapid tRNA decay pathway; T�CSL, T�C stem loop; TRAMP, Trf4/Air2/Mtr4p polyadenylation complex; tRNA, transfer RNA;VL: variable stem loop.
cmo5U34 and mcmo5U34 synthesis (Figure 2aand Table 1). Besides E. coli, a near-completedescription of tRNA modification genes hasbeen predicted using a comparative genomicapproach for only M. capricolum (35), but the
genes have not been experimentally validated.In eukaryotes, a bioinformatic analysis of theArabidopsis thaliana genome led to the iden-tification of 90 predicted modification genes(27), setting the stage for further experimental
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Table 1 Known pathways for complex modifications that require more than two enzymesa
Modifications Organismsb Known enzymes Commentsc ReferencesPosition 34mcm5U/ncm5U
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Table 1 (Continued )
Modifications Organismsb Known enzymes Commentsc Referencesm6t6A E. coli TsaA – (T. Suzuki & V. de
Crecy-Lagard,unpublishedresults)
ms2t6A B. subtilis IscS IscU MtaB – (5, 6)H. sapiens/M.musculus
Cdkl1 – (147)
i6A E. coli Yeast MiaA – (16)ms2i6A E. coli MiaB IscS IscU – (16)ms2i6o6A S. typhimurium MiaE – (16)yW S. cerevisiae Trm5, Tyw1,Tyw2,Tyw3,Tyw4 – (137, 156)o2yW H. sapiens +Tyw5 – (68, 103)imG derivatives Archaea Trm5a/b/c,Tyw1,Tyw2,Tyw3 – (114)Other positionsG+15 Archaea FolE QueD QueC QueE TgtA
(ArcS or Gat-QueC orQueF-like)
– (114, 116)
s2m5U54 T. thermophilus TrmFO – (101)s2m5U54 IscS or SufS, TtuA, TtuB, TtuC – (105)
Thiolation reconstitutedin vitro
(104)
aModifications that require two or fewer enzymes can be found at the Modomics database: http://modomics.genesilico.pl/.bArabidopsis thaliana, Bacillus. subtilis, Caenorhabditis elegans, Escherichia coli, Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Salmonella typhimurium,Schizosaccharomyces pombe, Thermus thermophilus.cBecause of constraints in reference numbers, Table 1 of Reference 117 and Table 1 of Reference 16 were used as a general source for the primaryliterature unless the work is posterior or not included in these tables.
work. A similar study was recently performedfor the human tRNA methyltransferase genes(133), but it needs to be expanded to all modi-fication genes. However, as information on thenature and position of tRNA modifications inHomo sapiens is scarce (19/49 tRNA species se-quenced), such an analysis will be quite chal-lenging. Examples of specific missing humantRNA modification enzymes include those re-sponsible for the formation of f5C34 in mito-chondrial initiator tRNA (15) and of acp3U20in tRNATyr
GUA (142). Finally, a full set of tRNAmodification genes was predicted in the ar-chaeon H. volcanii (55). These have been par-tially experimentally validated, and the currentstatus was recently reviewed (114). In sum-mary, a full description of tRNA modificationgenes in one model organism per kingdom is
nearly complete. Transferring this informationto other organisms will require special care forthe reasons discussed below. Such an endeavorshould be helped by the recent adaptation ofmass spectrometry and deep sequencing tech-niques toward the detection and quantificationof RNA modifications (23, 43, 48, 149, 155)that will allow researchers to identify and maptRNA modifications in many more organismsin the near future.
m1G/TrmDm6A/TrmN6m2A/?i6A/MiaAms2i6A, t6A, m6t6A/ see Table 1
S. cerevisiaeb
E. colia
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two enzymes in their biosynthesis pathwayare located at positions 34 and 37 of the ASL(Figures 1 and 2). The synthesis of some ofthese complex modifications, like mcm5s2U34in yeast cytoplasmic tRNAs, requires morethan 20 proteins (Table 1 and Figure 3).Deciphering these pathways has unearthednew chemistries (80, 88, 89, 113, 141), led tothe elucidation of the function of conservedand essential gene families (40, 41), and shownpotential links between tRNA modificationpathways and other cellular processes (25, 40).The synthesis of the most complex bases foundin tRNA, the imG derivatives such as yW(Table 1), is now fully elucidated and wasrecently extensively reviewed for yeast (137),Archaea (34, 114), and higher eukaryotes (68,103). Here the focus is on the synthesis of thio-lated modifications, xm5U34 (5-aminomethyl-uridine derivatives), and Q derivatives.
Complex Relay Chains Are Requiredto Thiolate Transfer RNAs
Sulfur atoms are found in several modified nu-cleosides (for a recent review, see 105). Thesesulfur elements are derived from cysteine afteractivation by a cysteine desulfurase (IscS inE. coli ) as an enzyme-bound persulfide (R-S-SH) (105, 122). The fate of the sulfur atomis different for each pathway but follows twomain routes that are either metal independentor metal dependent. In the metal-independentpath, the persulfide can be directly transferredto the tRNA targeting enzyme, such as theE. coli ThiI that catalyzes the formationof s4U8 (Figure 2a). The transfer of thepersulfide moiety can also be indirect,
requiring transits through a sulfur relay chainbefore transfer to the tRNA targeting enzyme.This is the case for the formation of s2U34derivatives in E. coli, where the sulfur moietypassages onto the TusA/TusBCD/TusE pro-tein relay before being transferred to the tRNAtargeting enzyme MnmA (105) (Table 1 andFigure 3a). In the metal-dependent route,the formation of an iron-sulfur cluster (Fe-S)through the Fe-S scaffold machinery (IscU inE. coli ) is required before transfer of the sulfurmoiety to specific tRNA modifying enzymessuch as MiaB in ms2i6A formation or TtcA ins2C32 synthesis (Figure 2a and Table 1). Inyeast, only the xm5U derivatives at position34 are thiolated (Figure 2b and Table 1), andthe thiolation machinery is more complex thanin E. coli with different thiolation pathwaysused for mitochondrial and cytosolic tRNAs(105) (Figure 3b). Both yeast pathways startwith the transfer of the sulfur moiety to thecysteine desulfurase (Nfs1) in the mitochondriarequiring Isd11 (83). In the mitochondrialpathway, the sulfur is transferred to Mtu1,a homolog of MnmA, through an unknownrelay protein (105) before directly modifyingcmnm5U34-containing tRNAs (Figure 3).In the cytosolic pathway, the sulfur moietyis transferred from Nsf1 to the relay proteinTum1 (83, 105). Tum1 may function as thesulfur shuttle between the mitochondrial andthe cytosolic compartments. In the cytosol, an-other cascade of sulfur transfer occurs throughthiocarboxylated intermediates involvingenzymes shared with the protein urmylationpathway (Uba4p and Urm1) before the transferof the sulfur moiety to the target tRNAs by theNcs2/Ncs6 complex (105) (Figure 3b).
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Known modifications and modification enzymes in Escherichia coli and yeast. (a) E. coli transfer RNA (tRNA).The corresponding genes are still missing for the modifications in red. Corresponding accession numbersand references can be found in the EcoGene database (http://www.ecogene.org/) (119; also see Table 1 ofReference 12 and Reference 16). (b) Saccharomyces cerevisiae cytoplasmic and mitochondrial tRNAs.Corresponding accession numbers and references can be found in the SGD database (http://www.yeastgenome.org/) (also see Reference 70 and Table 1 of Reference 117). Additional references include therecent discovery of TRM140 as responsible for Cm32 (32, 106).
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Figure 3Synthesis of 5-amino methyl-uridine derivatives (xm5U34) at position 34 in Escherichia coli and yeast.(a) Current model for the synthesis of mnm5s2U34 in E. coli (inspired by References 98 and 104) (formationof mnm5se2U34 is not shown, see Table 1). (b) Current model for the synthesis of cmnm5s2U34 in yeastmitochondria and of mcm5s2U34 and ncm5Um34 in yeast cytoplasm (24). R indicates the tRNA moleculesin the structures of the modified base. All enzymes are listed in Table 1.
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Synthesis of xm5U34 Derivatives
As discussed below, substitutions at U34are critical for correct decoding. The mostcomplex of these are the xm5U34 derivativeslisted in Table 1 (for a recent review, see14): The nm5U derivatives are found inbacteria and mitochondria, whereas the cm5Uderivatives are found in eukaryotes (Figure 3).The enzymes MnmE and GidA (MnmG)are involved in the formation of cmnm5U inbacteria (Figure 3a and Table 1). MnmE isthe archetype of guanine nucleotide-bindingproteins activated by nucleotide-dependentdimerization (GADs) (92), and GTPase ac-tivity is required for catalysis (93). GidA andMnmE form a α2β2 complex involving largeconformational changes (20, 92). The currentmodel is that the MnmE·GidA complex usesmethylene-tetrahydrofolate (THF) to formthe C5-methylene moiety and then catalyzesthe formation of cmnm5U when glycine isincorporated or of nm5U when ammoniumis incorporated (Figure 3) (93, 98). Thesereactions require FAD and NADH, and acatalytic mechanism has been proposed (98,123). In mitochondria, the pathway is probablysimilar to that found in bacteria, with the addedpossibility of incorporating taurine instead ofglycine in mammals to make τm5U (Table 1)(129). Subsequently, the FAD-dependentMmmC1 cleaves the glyoxylate moiety ofcmnm5U to form nm5U. In some bacteria,MnmC2 then catalyzes the SAM-dependentmethylation to produce mnm5U (Table 1 andFigure 3a).
The understanding of the biosynthesis ofcm5U34 derivatives in the eukaryotic cyto-plasm is incomplete and the latest model issummarized Figure 3b. Recently, componentsof the Elp complex were shown to be involvedin the formation of mcm5U and ncm5U(Table 1), but the pathway has not been re-constituted in vitro and the exact intermediateproduced is not clear (24). Methylation of thecm5U intermediate into mcm5U requires theSAM-dependent Trm9 and Trm122 enzymes(Table 1), but the Trm9/Trm122 complex may
also be involved in the previous steps (24). Inhigher eukaryotes, ALKBH8, the dioxygenaseresponsible for the formation of the hyper-modifications (S)-mchm5U and (R)-mchm5U(Table 1), can be a discrete protein as inA. thaliana or fused to the Trm9 domain as inmammals.
Synthesis and Salvage of QueuosineDerivatives in Bacteria and Mammals
Q is a modification found at the wobble positionof tRNAs with GUN anticodons in many bac-teria and eukaryotes (71). The hypothesis thatQ is derived from GTP was confirmed whenGTP cyclohydrolase I, the first enzyme of thefolate pathway, was also shown to be the firstenzyme of the Q pathway (115). Comparativegenomics methods combined with experimen-tal validation were used to identify three otherQ biosynthesis enzymes: QueD, QueE, andQueC (45, 118). QueD is the second enzymeof the Q pathway and catalyzes the formationof 6-carboxypterin (88, 89), whereas QueE andQueC catalyze the subsequent steps to producethe 7-cyano-7-deazaguanine (preQ0) interme-diate (89) (Figure 4). PreQ0 is also an interme-diate in the synthesis of archaeosine (G+) (111).G+ is found only at position 15 of archaealtRNAs; for a recent review of the archaeosinesynthesis pathway, see Reference 114. Inbacteria, preQ0 undergoes further reductionto 7-aminomethyl-7-deazaguanine (preQ1)by the NADPH-dependent enzyme preQ0
oxidoreductase (QueF) (141). The remainderof the pathway involves the insertion of preQ1
into the tRNA by the tRNA-guanine transgly-cosylase (bTGT) enzyme, the formation of oQby the enzyme QueA, and the final reduction ofoQ to Q by the recently discovered QueG, (seeFigure 4) (94; for a review, see 66). AlthoughQ is ubiquitous in eukaryotes and bacteria, onlybacteria are capable of de novo Q biosynthesis.Animals and plants acquire queuine (q), thefree base of Q, from diet and/or flora (74)(Figure 4). Eukaryotic TGT enzymes (eTGT)catalyze the insertion of q and, therefore, have
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P-P-P-O
OH OH
O
N
NHN
N NH2NH2N
O-P-P-P
OHO
HN
NH
N
OHNH2N
OH
O
HN
NH
HN C
O
NH
N
NH
N NH2
O
NH
NH
N NH2
OH2N
N
NH
N NH2
ONH3
tRNA
N
NH
N NH2
ONH2
tRNA
OHO
HO
N
NH
N NH2
ONH2
tRNA
HO
HO
NH
NH
N NH2
ONH2
HO
HO
O
GTP
FolE QueD QueEC
QueA bTGTQueG
H2NTP 6-carboxytetrahydropterinpreQ0
preQ1
preQ1-tRNAEpoxyQ-tRNAQueuosine-tRNA
Queuine (q)
Eukaryotes
Bacteria
THF
eTGT
Q/Q5’P?
tRNA
QueF
Figure 4De novo synthesis of queuosine (Q) in Escherichia coli and queuine (q) salvage pathway in eukaryotes. GTP is the precursor of Qsynthesis in bacteria, and the pathway shares a common step with the tetrahydrofolate (THF) pathway. Eukaryotic TGT (eTGT)inserts q in transfer RNA (tRNA). Both Q and Q-5′-phosphate (Q5′P) can be salvaged (58, 76), yet the salvage pathway and the specificnucleosidases required to liberate the q base have not been characterized. Further modification to GalQ and ManQ of tRNATyr andtRNAAsp, respectively, in mammals and to GluQ of tRNAAsp in bacteria is not shown (Table 1). Abbreviation: H2NTP,dihydroneopterintriphosphate.
different specificity from bacterial TGTs thatfavor preQ1 over q (Figure 2) (28, 128). Inmammals, the TGT subunit (or QTRT1)and the Qv1 subunit (or QTRTD1) form anactive heterodimer even if the catalytic residuesreside in the TGT subunit per se (21, 29). Theeukaryotic q salvage pathway remains to beelucidated.
VERSATILITY OF TRANSFER RNAMODIFICATION ENZYMES:SEVERAL SOLUTIONS FOR THESAME PROBLEM
A challenge in the tRNA modification field isthe inability to predict reliably the presence orabsence of a specific modification based on thepresence or the absence of a homolog of an
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experimentally characterized tRNA modifica-tion gene. Indeed, members of the same proteinfamily can catalyze different reactions in differ-ent organisms. Also, the same modification canbe introduced by different enzymes in differentorganisms as cases of convergent evolution oc-cur repeatedly in the tRNA modification arena.These issues are discussed below.
Methylation of m5U54 in BacterialTranfer RNAs Can Be Catalyzed bySAM-Dependent orFolate-Dependent Enzymes
The m5U modification (also called rT) atposition 54 is present in most analyzed bacteriawith the exception of Mycoplasma capricolum(71). The modification is introduced in E. coli bythe SAM-dependent methyltransferase TrmA(Figure 2a). However, no TrmA homologscould be identified in other organisms knownto carry this modification, such as Bacillussubtilis. This discrepancy was resolved with thediscovery of TrmFO, a flavoprotein unrelatedto TrmA that uses N(5),N(10)-methylene-tetrahydrofolate as methyl donor to modifyU54 (136). The phylogenetic distribution ofmembers of the TrmA and TrmFO familiesshow an inverse correlation, and one or theother is found in most sequenced bacteria (136).
An Archaeal Homolog of a BacterialRibosomal RNA MethyltranferaseMethylates Tranfer RNA
Found in the hyperthermophilic ArchaeaPyrococcus abyssi, PAB0760 and PAB0719 aretwo members of the RlmD family, which inE. coli catalyzes the formation of m5U at posi-tion U1939 of 23S RNA (86). However, nei-ther of these enzymes catalyzes the reactionnormally attributed to RlmD family members.PAB0719 introduces m5U54 in tRNA (135),and PAB0760 introduces the m5U modificationat another position in 23S RNA (correspondingto position 747 in E. coli ) (9). This illustrates thedifficulty of inferring functional annotations fortRNA modification proteins from one organ-ism to another using similarity scores alone.
MiaB Homologs Can MethylthiolateTransfer RNAs or Proteins
Methylthiolations of tRNAs are catalyzed byradical-SAM enzymes of the MiaB family. Thefirst member of this family to be experimentallycharacterized, MiaB, catalyzes the formationof ms2i6A37 in E. coli tRNAs (Figure 2a).Analysis of the sequences and distributionof MiaB homologs in sequenced genomesrevealed that the MiaB family can be separatedinto four subgroups (72; reviewed in 8). Thecanonical MiaB subgroup codistributes withMiaA, the enzyme involved in the formationof the MiaB substrate, i6A37-containing tRNA(Figure 2a), and is found in most bacteria andin many eukaryotes [with the exception of yeastthat harbors only i6A in tRNA (Figure 2b)].The substrates for the other subgroups haverecently been identified. The YqeV (or MtaB)subgroup is involved in methylthiolation of t6Ain bacteria such as B. subtilis (but is not foundin E. coli ) (8). Members of the CDKAL1 (oreMtaB) subgroup catalyze the same reaction ineukaryotes and Archaea (8). Finally, the YleGor RimO subgroup found in many bacteria andeukaryotes does not methylthiolate a nucleo-side in a tRNA molecule but instead targets anaspartic acid in ribosomal protein S12 (8).
The Universal Modification t6A IsSynthesized in Different Kingdomsby Divergent Pathways Sharing aCommon Core
Along with m1G37, t6A37 is one of the rareuniversal modifications of the ASL. The E. colit6A biosynthesis pathway was recently reconsti-tuted in vitro and requires four different proteincomponents (36). Of these, TsaC and TsaD arepart of the universally conserved YrdC/Sua5and YgjD/Kae1/Qri7 families that have alsobeen implicated in t6A synthesis in yeast (40,41, 127), whereas TsaE and TsaB are membersof the bacteria-specific protein families YjeEand YeaZ, respectively. The synthesis pathwaysin eukaryotes and Archaea are still not fullyunderstood, as homologs of YjeE and YeaZ are
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missing outside the bacterial kingdom and Sua5and Kae1 alone are not sufficient to synthesizet6A in the cytosol (127). In eukaryotes andArchaea, Kae1 is a member of the KEOPS(kinase, endopeptidase, and other proteinsof small size) complex. This complex is in-volved in different processes, such as telomerehomeostasis, transcription, DNA integrity,and chromatin remodeling (for reviews, see 40,108). Other subunits of the KEOPS complexcould be required for t6A formation in additionto Kae1 (33, 127). t6A synthesis may alsooccur through another pathway in organelles;the mitochondrial-targeted protein Qri7 is afunctional homolog of TsaD (40, 108, 127), butthe rest of the t6A mitochondrial machineryhas not been characterized. In summary, thet6A biosynthesis pathway is only partiallyconserved, with a set of core enzymes and withadditional kingdom (or organelle-)-specificvariations.
DIRECT ROLES OFMODIFICATIONS IN TRANSFERRNA FUNCTION
The recent characterization of most tRNAmodification genes in model organisms (dis-cussed above) has allowed researchers to probethe role of tRNA modifications in vivo. Thecommon view is that tRNA modifications havemainly direct or primary roles in translation,as discussed in this section. However, the ideathat tRNA modifications (and their pathways)can take on regulatory functions has recentlyreemerged. The more complex or second-orderfunctions (discussed in the next section) coverthe newly discovered roles of tRNA mod-ifications as connectors between translation,metabolism, and stress response (Figure 1).
Most and Foremost: A Central Roleof Transfer RNA Modificationsin Decoding
The 20 universal proteogenic amino acids areencoded by 61 codons (64 total minus thethree stop codons), and most are organizedin degenerated codon family boxes in which
synonymous triplets code for the same aminoacid. There are eight unsplit boxes (such as Leu)in which all codons code for the same aminoacid, five two-split boxes (such as Asp/Glu) inwhich the two purine ending codons encodefor one aminoacid and the two pyrimidineending codons encode for another, and threespecial codon boxes (Ile/Met, Tyr/Stop, andCys/Stop/Trp). The fact that most amino acidsare encoded by more than one codon is calleddegeneracy. Decoding of the genetic code relieson the interaction between the three bases ofthe mRNA’s codon triplet (numbered 1, 2, and3) and the three anticodon bases of the cognateaminoacyl-tRNA (numbered 36, 35, and 34 inthe anticodon stem loop) (Figure 5). Althoughthe standard rules of the Watson-Crick pairing(A/U, U/A, G/C, C/G) strictly govern theinteraction between base pairs 1/36 and 2/35,the 3/34 base pairing can be nonstandard(wobble interaction). Therefore, one tRNAmolecule can often decode several codons.Nucleoside modifications ensure that thedecoding process is stringent enough todiscriminate between closely related codons,a problem found mostly in two-split andspecial boxes, and yet relaxed enough to allowdecoding of more than one codon. Differ-ent organisms use distinct but convergentstrategies to optimize decoding by modifyingspecific tRNAs, predominantly at position 34of the ASL (which interacts with the thirdcodon base via non-Watson-Crick base pairingor wobble base pairing) (Figure 5b). Strategiesdiffer depending on whether a given codon ispart of a four-unsplit codon box in which fourcognate codons coexist or part of a two-splitcodon box containing two pairs of codons eachcoding for a different amino acid. The mostcommon strategies are summarized below andin Figure 5a, but a more complete descriptioncan be found in Reference 54.
In two-split codon boxes, modificationsare used to better discriminate between thecognate pyrimidine-ending and noncognatepurine-ending codons (Figure 5a). In botheukaryotes and bacteria, the pyrimidine-endingcodons are generally read by a tRNA harboring
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A
A
G
U
C
A
G
G
U
C
U U
C
A
C
G
U
C
[E]I
[A,B]G
[B]U*
Q and Q# G**
Cm
[E]I
[A,B]G
C
C
Wobble base
tRNA
ASLGm
A
G
A G U C
[B]xmn5U
[E]xcm5U
[A]?U
C C
[B]xo5U
[E]ncm5U
[B]U*[B]xo5U
[E]U
[E]I
[A,B]G
[B]xo5U
[E]ncm5U
[B]U*
[E]I
[A,B]G
[E]Ψ[B]k2C***
[A]agm2C***
[E]I
[A,B]G
[B]xo5U
[E]ncm5U
[B]U*
[B]U*
[B]U*
[E]I
[A,B]G
[B]xo5U
[E]ncm5U
C
C
[E]I
[A,B]G
[B]xo5U
[E]ncm5U
C C C C
C C
C C
[B]U*
[B]I
Q
[B]xmn5s2U
[E]xcm5s2U
[A]?U
Q
[B]xmn5s2U
[E]xcm5s2U
[A]?U
[B]xmn5s2U
[E]xcm5s2U
[A]?U
Q
[B]xmn5Um
[E]xcm5Um
[A]?U
G
[B]xmn5U
[E]xcm5U
[A]?U
G***
[B]mnm5U
[E]mcm5U
[E,B]I
[A]G
Two-split boxes
Special boxes
Unsplit (U) boxes
Ile codon (AUA)
Phe
Ser
Tyr
Leu StpStp
Trp
Leu Pro
His
Arg
Gln
Ile
Thr
Asn
Lys Arg
iore
Met
Val Ala
Asp
Gly
Glu
5'
Ser
Cys
3'
3' 5'
Anticodon
34 36
34
Anticodon 35
35 36
Codon
mRNA
3rd 2nd 1st
ba
Figure 5All transfer RNA (tRNA) decoding strategies depend heavily on modifications at position 34 of the tRNA. (a) Anticodon modificationsused in decoding. This table compiles the major strategies that organisms have evolved to accurately and efficiently decode their geneticmaterial with an emphasis on tRNA anticodon position 34, which is heavily modified mainly to ensure discrimination between purinesand pyrimidines within two-split or special boxes ( yellow); unsplit (U) codon boxes are also represented. Each row corresponds to adifferent tRNA, and distinction is made depending on the kingdom considered. When the row is empty, the tRNA is not modified atthat position and an isoacceptor tRNA decodes the corresponding codon. s2 indicates that se2 can be found instead. Single asteriskrepresented unmodifed U but presence of G or C at position 35 and /or 36 as well as other elements in the anticodon stem loop (ASL)such as C at position 32 are required. (b) Double asterisks indicate the G34 is unmodified but is flanked by a modified C32, e.g., � inEscherichia coli tRNA Cys and s2 in E. coli tRNASer. (c) Triple asterisks indicate the tRNAIle
CAU has a Met (CUA) anticodon but C34 ismodified to lysidine in bacteria and to agmatidine in Archaea to decode specifically the Ile codon (AUA). C indicates the C34 sparingstrategy never applies in these two cases (i.e., there is always a tRNA with a C34, modified or not, to decode the corresponding codon).A gray-shaded C denotes a possible C sparing strategy depending on the organism. In some organisms, Trp belongs to a two-splitcodon box where UGA is assigned to Trp instead of stop and is read by a tRNA Trp with a UCA anticodon with a modified U34 thatpairs with purines (tRNATrpCmCA may or may not be spared and termination occurs only at UAA and UAG). Q# indicates Q andderivatives (see Figure 4 and Table 1). In eukaryotes, the U35 of tRNA Tyr is always modified to �. i,eMet: In all organisms, theC34AU anticodon is harbored by two disinct tRNA Met encoded by different genes, one for the regular elongator eMet and one for theinitiator iMet. In rare cases the Met/Ile box can be a two-split codon box. In human mitochondria, for example, both AUA and AUGare read as methionine with a tRNA harboring a C34 that can be modified to f5C. Abbreviations: A, Archaea; B, bacteria; E, eukarya(mainly yeast).
a modified G at position 34 (except for Cys andSer). G34 is often modified to the Gm or Q (andQ derivatives) wobble pairing with U3 or C3.The purine-ending codons are read either by asingle tRNA carrying a modified U at position
34 (reading A3 or G3 and mostly used by bacte-ria) or by two tRNAs, one harboring a modifiedU and one harboring a C (generally unmod-ified) (reading G3). U34 is often modified toderivatives of xnm5U in bacteria and xcm5U in
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eukaryotes (reading A3) but can also be doublymodified for added specificity. The oxygen atposition 2 of the uracil ring is then replaced by athio or seleno group (cmnm5s2U or mnm5se2U)or a methyl group is added on the 2′-hydroxylof the ribose (cmnm5Um or ncm5Um).
In four-unsplit codon boxes, the simpleststrategy is found in Mycoplasma and mitochon-dria where a tRNA harboring an unmodifiedU34 and a G or C in position 35 and/or 36(among other requirements) can pair with U3,C3, A3, and G3 (35) (Figure 5a). In all othercases described so far (with the exception of theArg and Leu boxes of specific organisms; see theend of this section), U34 is generally modifiedto read A3 or G3 (to xo5U, xcm5U or xnm5U), asin the two-split boxes, and coexists with isoac-ceptor tRNAs usually carrying a G34 or I34 todecode U3 and C3 or in some instances a C34to decode G3 (Figure 5a).
Modifications at position 34 contribute totranslation fidelity by ensuring codon discrimi-nation by tRNAs. This issue is especially crucialin the Ile/Met, Cys/Stop/Trp, and Tyr/Stopboxes (Figure 5a). In the Ile/Met box, Met isencoded by one single AUG codon, whereasIle is encoded by AUA, AUU, and AUC. Thismeans that a tRNA capable of distinguishingbetween the two purines at the wobble base isrequired. In eukaryotes, the minor tRNAIle
�A�
reads AUA, whereas a tRNAMetCAU with
C34 modified to ac4C or Cm (depending onthe organism) is used to selectively decodeAUG (Figure 5a). The modification of C34is thought to strengthen the C:G base-pairinteraction and thus prevent misreading of theAUG Met codon. In bacteria, the AUA codonis read by a tRNAIle
k2
CAU. The k2C modifica-tion is unique in that it changes the amino acididentity of the tRNA from Met to Ile (125). Asimilar solution occurs in Archaea but with aslightly different modification, agm2C (64, 87).
Finally, in the Cys/Stop/Trp box, the uniqueTrp codon UGG is accurately distinguishedfrom the UGA stop codon recognized by a re-lease factor (for a review, see 100) by modifica-tion of the C34 of tRNATrp to Cm, whereas inthe Tyr/Stop box, G34 modification to Q or Q
derivatives to read U3 and C3 allows discrim-ination between the Tyr codons (UAU/UAC)and stop codons (UAA/UAG) (Figure 5a).
Position 37, which is on the 3′ side of theanticodon (also called the dangling base) isalso often modified. As a rule, when position36 is an A or U, position 37 is modified. Thisdiverse set of modifications (Table 1 andFigure 2) mainly stabilizes the first base pairof the codon/anticodon interaction, especiallyA:U and U:A pairs, and thereby contributesto accurate decoding by reducing frameshifts(57). These modifications are all thought tobring order to the ASL structure by preventingintraloop base pairing, thereby maintainingthe ASL in an open loop structure that allowsproper codon-anticodon binding (1).
Transfer RNA Modifications Insideand Outside the Anticodon Stem LoopAre Important for MaintainingTransfer RNA Structure
It is well established that tRNA molecules foldinto the so-called L shape (Figure 1) and thatthe major interactions maintaining the L shapeoccur at the elbow of the tRNA molecule,where the D loop (containing the modifica-tion dihydrouridine) and the T�C loop (con-taining thymine, or m5U, and � modifications)meet (46). Nucleoside modifications participatecollectively in the stabilization of the tRNAmolecule in vivo, and some of their impor-tant structural functions include restriction ofnonfunctional alternative folding, cooperativebinding of Mg2+, and thermal stabilization.
Methylations are the simplest and mostfrequent modifications found in tRNAs, andthey can occur at every position of the targetnucleotide (97). Methylations destabilizeWatson-Crick interactions and lead to largestructural changes in the global tRNA fold(1, 62). m1A9 in human mitochondrial tRNALys
is a classic example of a methylation that affectstRNA structure, as the methylation displacesthe structural equilibrium from an alternativehairpin structure to the functional cloverleafstructure (96). Other known modifications with
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critical structural roles are the � at positions32 and 39 that shape the ASL (96).
Mg2+ and polyamines are important forproper tRNA folding and structure stabiliza-tion (56, 121), and their modifications areimportant players in RNA-Mg2+/polyamineinteractions. For example, m5C in yeasttRNAPhe
GAA enhances Mg2+ binding (30, 75,157), and branched pentamines inhibit m5Cformation in specific tRNAs (61). Severalmodifications such as ac4Cm, m1Im, �, Gm,and m2
2Gm are involved in thermal stabiliza-tion of tRNA (reviewed in 62, 96), which isparticularly relevant in the following: (a) hy-perthermophiles that modify their tRNA moreextensively when grown at higher temperature(107) and contain doubly modified nucleosidessuch as ac4Cm or m1Im and (b) unique modi-fications such as s2T (63). High-temperaturesurvival of Thermus thermophilus is strictlydependent on the formation of s2T (124).Finally, genes involved in the biosynthesis ofmnm5s2U and of five thiolated bases in tRNAwere recently shown to be required when E.coli was exposed to high temperatures (99).
Modifications as IdentityDeterminants or Antideterminantsfor Specific Recognition of TransferRNA Molecules
Every tRNA isoacceptor is fine-tuned to bespecifically recognized by its cognate AARSand modifications can have very importantroles in this process. Ten modified nucleosideshave been characterized as identity determi-nants for AARSs (for a review, see 47). Thesemodifications are all located in the ASL andincrease the aminoacylation reaction efficiencyby improving either kcat or Km or both. Thecatalytic efficiency of an AARS is expressed inμM−1 s−1 and is equivalent to the ratio kcat/Km,where the kcat value is the catalytic rate constant(s−1) and the Km value is the affinity constant(μM) for the tRNA substrate. These kineticparameters can be derived from the doublereciprocal plot: initial rate−1 = f ([tRNA]−1)also known as Lineweaver-Burk plot. (47).
k2C34 in E. coli tRNAIleCAU and m1G37 in yeast
tRNAAspGUC are antideterminants and prevent
tRNA recognition by E. coli methionyl-tRNAstynthetase and yeast arginyl-tRNA synthetase,respectively (47).
Other components of the translation appa-ratus require the presence of modifications torecognize tRNAs. m5U54 affects the structureof the tRNA T�C loop, changing the bindinginterface contacts and thereby altering recogni-tion by EF-Tu (39). Some tRNA modificationenzymes require prior modifications, establish-ing a chronological order in the introductionof modifications (96). For example, formationof the s2T54 modification in T. thermophilusis dependent on the presence of m1A58 (65).Similarly, Gm18 and m1G37 are reduced inT. thermophilus cells lacking m7G46 in tRNAPhe
(132). tRNA thiolations can also act as an an-tideterminant in tRNA editing. Specifically,mitochondrial tRNATrp in Trypanosoma bru-cei takes two forms: the nuclear-encodedtRNATrp
CCA that reads UGG codons and anedited version of tRNATrp
UCA that reads UGAcodons (3). Lack of thiolation of U33 increasesthe level of C to U editing (152). Finally,maturation of tRNAs can be very complex withspatial separations of the different steps. Forexample, in yeast pre-tRNAPhe
GAA is exportedfrom the nucleus and then spliced and partiallymodified in the cytoplasm (110). The nonfullymodified (or apomodified) tRNA is then im-ported back into the nucleus where the m1G37modification is introduced by Trm5. The tRNAis then exported back to the cytoplasm wherethe yW machinery finishes the maturationprocess (110). tRNA modifications are alsorecognized by RNA surveillance pathwaysas checkpoints for the structural/functionalintegrity of both pretRNA and maturetRNA molecules. In S. cerevisiae (and prob-ably in higher eukaryotes), the TRAMP(Trf4p/Air2p/Mtr4p polyadenylation) com-plex recognizes aberrant pre-tRNAi
Met
transcripts lacking m1A58 and targets them for3′–5′ degradation by the nuclear exosome andRrp6 (for a review, see 117). By contrast, theRTD (rapid tRNA decay) system (exonucleases
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RAT1, Xrn1, and Met22) acts selectivelyon mature tRNAs lacking certain pairs ofmodifications and targets the tRNAs for 5′–3′
degradation (117). Mature tRNAValAAC lacking
both m5C and m7G46 (7-methylguanosine)and mature tRNASer
CGA and tRNASerUGA
lacking both Um44 (2′-O-methyluridine) andac4C12 (4-acetylcytidine) are rapidly degradedat 37◦C. Therefore, the corresponding doublemutants lacking both modification genes showa temperature-sensitive growth phenotype(2, 78). Because the structural integrity ofthe T stem is the major tRNA recognitiondeterminant for the RTD system, the contri-bution of the modifications is probably indirectthrough their role in modulating the stabilityof the tRNA tertiary structure (150).
Translation Phenotypes Causedby a Defective Transfer RNAModification Machinery
Numerous studies have tackled the delicatetask of understanding the individual contri-butions of specific modifications to transla-tion accuracy and efficiency, whether in vitro(using apomodified tRNA and ASL as sub-strates) or in vivo (using deletion mutants andreporter systems). Lack of several modifica-tions such as Q34, mnm5s2U34, ms2i6o6A37,m1G37, yW37, and t6A37 has been associatedwith increased frameshift phenotypes (40, 138,145). The generally accepted model is that nu-cleoside modifications, mainly those involvingpositions 34 and 37, contribute to accurate de-coding by ordering the ASL and stabilizing thecodon-anticodon interactions, therefore pre-venting ribosome pausing and slippage of thepeptidyl-tRNA (7, 59, 69).
Additional modifications have been asso-ciated with translation initiation defects. Forexample, S. cerevisiae strains lacking t6A37 inthe cytoplasm display increased leaky scanning(84) and increased misinitiation at GUGcodons (40). This modification is required foraccurate start-codon selection in Eukaryoticcytoplasm, in part by restricting tRNAi
Met
decoding capacity to the single initiation
codon AUG (77, 82). Another modificationthat affects initiation is f5C34. In humanmitochondrial tRNAMet
CAU, the f5C34 mod-ification unlike the t6A modification in theexample mentioned just above expands codonrecognition from the traditional AUG to AUA,AUU, and AUC, thereby contributing toaccurate initiation codon selection this time inorganelles (15). Base modifications within or 3′
to the anticodon can also impact the efficiencyof translational stop-codon read-through bynatural nonsense suppressor tRNAs (reviewedin 11), an observation proven by tRNAmutagenesis studies (see, for example, 154).
COMPLEX PHENOTYPESASSOCIATED WITH THELACK OF TRANSFER RNAMODIFICATION
Because of their roles in tRNA structure andfunction described above, the absence of oneor more tRNA modifications presumably cre-ates an error-prone translational system. In afew rare cases, this leads to cell death. In gen-eral, however, cells are able to cope with poolsof diverse and/or erroneous proteins. Indeed,the absence of most modifications has minimaleffects on growth under standard conditions.Why then are modifications so conserved andso much energy allocated to their synthesis?Recent work has shown that the role of thesemodifications is revealed under specific circum-stances: given interactions with a host, understress, or if another protein is missing.
Importance of Transfer RNAModifications for Cell Growth
Nucleoside modifications affect the tRNAlifecycle at most stages (as discussed above).However, only few modifications are strictlyessential in vivo: I34 in both bacteria and yeast(153), k2C34 in bacteria and agm2C in Archaea(64, 125), t6A37 in prokaryotes (40, 41), �
54/55 in Archaea (19), as well as m1A58 inyeast (4) and thermophilic bacteria (124). Theabsence of few other modifications can confer
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severe growth phenotypes such as that oft6A37 in yeast (40, 41) or m1G37 and xm5U34derivatives in bacteria and yeast (17; reviewedin 117). These defects can be caused by essen-tial roles in decoding such as that of k2C34 inbacteria (see above) or in stabilizing tRNAi
Met
(4). Often, however, the cause of the growthphenotype is not known. It has been postulatedthat the high translation errors caused by theabsence of a specific modification, such as thehigh frameshifting observed in the absenceof m1G37 (17, 138), could affect growth, butthe strict cause-and-effect relationship hasnever been proven. The incorrect translationof specific proteins, not a general translationdefect, may be the cause of some of the growthphenotypes.
Importance of Transfer RNAModification for the Translationof Specific Proteins
Because modifications affect codon-anticodoninteractions at specific codons, it was postulatedin the early 1970s (148) that changes in mod-ification levels may modulate the expression ofspecific proteins, sometimes leading to observ-able phenotypes. A classic example is the expres-sion of the Shigella virulence gene virF, whichis dependent on the presence of Q34 (38). A Q−
mutant that does not show any growth defectin a test tube is less virulent than the wild-typestrain. A more recent example emerged fromthe discovery of the molecular basis for mito-chondrial diseases caused by tRNA modifica-tion defects (reviewed in 129). Indeed, MELAS(mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes) is causedby a mutation in mitochondrial tRNALeu
UUR
that eliminates the naturally occurring taurinemodification. The A3243G and T3271C pointmutations of mitochondrial tRNALeu
UUR resultin tRNAs lacking τm5U34, which in turnresults in considerable reduction of UUGdecoding. Although UUG usage is relativelylow in most proteins, eight are found in thegene encoding ND6, which is a component ofrespiratory chain complex I. In addition, a point
mutation (A14453G) in the ND6 gene wasassociated with severe MELAS syndrome. Thisis the first example of a link between a decodingdefect caused by lack of a specific tRNA mod-ification and a gene expression defect (ND6deficiency leading to complex I deficiency),which was finally linked to a clinical phenotype(MELAS). Several other examples will surelyfollow as mutations in the enzyme responsiblefor methylthiolation of t6A (Cdkal1 in H.sapiens) (Table 1) have recently been shown tolead to type 2 diabetes. It has also been proposed(but not proven) that synthesis of proinsulinis affected in Cdkal1-deficient individuals(147).
Phenotypes Linking ModificationEnzymes to Pathways Other thanTransfer RNA Maturation
There are several cases in which null mutantsof tRNA modification genes display lethalor severe pleiotropic phenotypes (see sectionabove). These cases prompt several questions:Are the phenotypes due to the absence of themodification in tRNA per se and, thus, due to atranslation defect? If so, the identification of theprotein(s) affected by the translation defects ispivotal. Are the phenotypes due to the absenceof the modification in a target molecule otherthan tRNA (another RNA or a protein)? Ordo modification enzymes harbor moonlightingfunctions not related to modification, as al-ready observed for several tRNA modificationenzymes (for examples of such, see 18)?
The urmylation pathway is a clear exampleof a tRNA modification pathway with twofunctions, each involving distinct cellularmechanisms. C-terminal glycine thiocarboxy-lated Urm1 serves both as a sulfur donor forthe thiolation of U34 (Figure 3b and Table 1)and as a protein modifier under oxidative stressin yeast and humans (49, 50, 140). As expected,urm1 defects are associated with pleiotropicphenotypes. Interestingly, some urm1 defectsare redundant with those of elp-defectivemutants. The Elp complex, composed of sixdifferent proteins subunits (Elp1 to 6), was first
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identified for its role in transcription as an H3and H4 histone acetylase but has since beenshown to participate in at least four distinctcellular processes: transcriptional elongation,polarized exocytosis, telomeric gene silencing,and formation of modified wobble uridines intRNA (for reviews, see 117, 143). However,as all elp phenotypes are suppressed by simul-taneous overexpression of tRNA isoacceptorsnormally harboring the mcm5s2U34 modi-fication, and the severity of the phenotypescorrelates with the amount of this modification(25), these could all be the consequence oftranslation defects. Sir4 (a known regulator oftelomere maintenance) has been identified asa candidate for a mistranslated protein causingsome of the elp mutants phenotypes (25).
The KEOPS/ECK complex case is verysimilar to the ELP situation. This complexcomprises several subunits conserved in eu-karyotes and Archaea (Kae1, Bud32, Cgi121,Pcc1, and Gon7 in yeast). Like ELP, thiscomplex was implicated in different processessuch as telomere homeostasis, transcription,DNA integrity, chromatin remodeling, and,more recently, tRNA modification (for reviews,see 40, 108). The recent discovery that Kae1is involved in t6A37 synthesis suggests that allthe observed phenotypes of KEOPS-defectivestrains could be due to translation defects(40, 127). Indeed, Sua5, the other knownenzyme involved in t6A37 biosynthesis, is alsoinvolved in telomere maintenance (91). Assome tRNAs (such as Lys, Thr, and Arg) carryboth mcm5U34 and t6A37, the presence ofone may be required for the synthesis of theother, possibly explaining some overlap in thephenotypes observed when subunits of boththe KEOPS and ELP complexes are deleted.Finally, bacterial t6A37 biosynthesis enzymesare all essential, but the molecular basis for thisphenotype remains unknown. Several attemptsto link bacterial t6A37 to cellular processesother than tRNA modification have failed togive a unifying view, with reported roles in cel-lular processes as diverse as DNA maintenance,membrane and cell-shape homeostasis, celldivision, and protein glycation (13, 60, 73, 108).
A REEMERGING REGULATORYROLE FOR TRANSFER RNAMODIFICATIONS?
tRNAs are at the heart of the translationmachinery. Thus, researchers were quick torecognize that synthesis, posttranscriptionalmodification, and degradation of tRNAs wouldbe highly regulated and integrated with thecellular response circuitry (18, 112, 151). In-deed, the levels of several modifications dependon growth rate, oxygen levels, or the presenceof vitamins or metals (18, 112, 151). Recentstudies strengthen the potential roles of tRNAmodifications in the cellular response to envi-ronmental stimuli, even if the mechanisms un-derlying these roles remain elusive (131, 155).
Local Codon Usage: A Built-InRegulation Strategy
Investigators have reported examples ofincreased bias for certain codons decodedby tRNAs with specific ASL modifications,potentially leading to modification-dependenttranslation efficiency of key regulatory pro-teins. The translation levels of the yeasttelomerase regulator Sir4 is dependent onthe presence of the ELP complex enzymesand, hence, of xm5U derivatives (25). Thetranslation levels of the yeast ribonucleotidereductase enzymes Rnr1 and Rnr2 are depen-dent on the presence of Trm9 and, hence, ofmcm5U (10). The corresponding genes areenriched in stretches of codons decoded by thecorresponding xm5U-containing tRNAs (10,25). Codon-usage analysis on whole proteomessuggests that the expression of hundreds ofproteins could be regulated in a similar fashion(10, 25, 40). This begs the question: Are thesestochastic events or are they revealing anunderlying regulation cascade?
Transfer RNA Modification Profiles:Varying Responses to EnvironmentalCues
tRNA modification profiles are influenced byparameters such as growth temperature or
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growth rate. For example, when thermophilesare grown at high temperature, their tRNAmodifications are increased (107). Also, thedegree of thiolation of individual tRNA speciesin E. coli varies dramatically with growthrates (42). Recently, it was shown that yeastexposure to chemicals such as H2O2 or MMS(methylmethanesulfonate) is concomitant withan increase or decrease of specific sets oftRNA modifications (10, 23). As trm9-deletionmutants are more sensitive to MMS exposureand, as stated above, the full expression ofkey proteins needed for survival under DNAdamage stress such as Rnr1/2 requires Trm9,it is tempting to postulate that this proteinis at the center of a regulatory cascade (10).However, the expression and/or activity ofthe Trm9 protein does not vary under thestress conditions tested (10), so one cannot yetconclude that the Trm9-dependent increasein the translation of specific stress-responseproteins is a regulatory mechanism.
Transfer RNA Cleavage in Responseto Stress
A newly discovered component of the eu-karyotic stress response is the endonucleolyticcleavage in the ASL region of a small fractionof the population of mature tRNAs (130, 131).These cleaved tRNAs are thought to inhibittranslation by different mechanisms, suchas functioning as small interfering RNAs ormicroRNAs, guiding cleavage of mRNAs, andforming repression complexes together withother factors (132). The ASL region is usually
heavily modified, so the question of a potentialrole for modifications as determinants orantideterminants of endonucleases naturallyarose. Such is supported by the previous find-ings that xm5U modifications are required forcleavage of target tRNA by the Kluyveromyceslactis γ-toxin (85) and that in Drosophila them5C38 modification introduced by Dnmt2protects specific tRNAs from stress-inducedcleavage, firmly linking tRNA modificationswith the tRNA cleavage mediated stressresponse (120).
CONCLUSIONS
The genomic revolution and the improvementof analytical tools have led to a renaissance ofthe tRNA modification field. The complexityand diversity of tRNA modification pathwaysremains astonishing; it includes some of themost elaborate pathways described in cellsto date. This complexity, which is not fullyunderstood, is a tribute to their importance inthe cell. The focus of this review is on tRNAmodifications, but modifications of rRNA alsoplay key roles in translation and use evolution-arily related enzymes (109). The synergismand redundancy between tRNA and rRNAmodifications remain unexplored. With therecent discovery of modifications in other typesof RNA not directly involved in translation,such as microRNAs or small interfering RNAs(43, 144), the knowledge accumulated onsynthesis and function of tRNA modificationswill likely transfer to other fields.
SUMMARY POINTS
1. A near complete characterization of tRNA modification enzymes in one model organismper kingdom has been achieved. This allows investigation of the function of modificationsin vivo
2. Very few tRNA modifications are essential, but many affect decoding and tRNA structureand stability
3. tRNA modifications can be checkpoints for tRNA integrity.
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4. Newly discovered tRNA modification enyzmes perform new chemistries.
5. The absence of specific modifications, particularly in the anticodon region, can lead tocomplex phenotypes.
6. RNA modification enzymes are difficult to annotate because transferring function bysequence similarity is often misleading.
7. Human diseases have been linked to the absence of tRNA modifications.
8. With more sensitive analytical techniques, researchers have been better able to detectand quantify modified nucleosides.
FUTURE ISSUES
1. The role of tRNA modifications as regulatory mechanisms needs to be studied further.
2. Additional work needs to focus on the emerging importance of tRNA modifications indiseases.
3. Researchers need to determine tRNA sequences in relation to the nature and positionsof modifications in more organisms.
4. Many of the in vivo roles of tRNA modifications remain undisclosed.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
M.B. is currently affiliated with the Department of Biochemistry at Emory University in At-lanta, Georgia. This work was supported by the National Institutes of Health (grant number R01GM70641 to V. de C.-L.). M.B. is recipient of a postdoctoral fellowship from the Human FrontierScientific Program. The authors thank Henri Grosjean, Patrick Thiaville, and Caroline Kohrerfor critical reading of the manuscript. We apologize to our tRNA modifications colleagues if wehave omitted their work, as this review required us to make difficult choices as a result of spaceconstraints.
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