Methylated nucleosides in tRNA and tRNA methyltransferases

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.


INTRODUCTION
The first tRNA sequence was determined in 1965 and numerous modifications were identified at various positions within the sequence (Holley et al., 1965). At almost the same time, several tRNA methyltransferase activities were detected in Escherichia coli cell extract (Hurwitz et al., 1964), which suggested that diverse enzymes are involved in tRNA modification. To date, more than 90 modified nucleosides have been identified in tRNA (Machnicka et al., 2013). Thus, the majority of modified nucleosides that have been discovered in different RNA species are found in tRNA. In the twenty-first century, the major modification pathways of tRNA have been elucidated on the basis of genome sequence data. These studies have demonstrated that the pathways of tRNA modification show diversity among living organisms. In this review, I focus on the methylated nucleosides in tRNA, together with tRNA methyltransferases, and introduce their basic roles as well as their more complex functions.

THE PRIMARY ROLE OF tRNA MODIFICATIONS IS THE REGULATION OF PROTEIN SYNTHESIS
Transfer RNA is an adaptor molecule that enables the genetic code of nucleic acids to be converted to amino acids in protein.

Trm5
The m 1 I37 modification is often observed in eukaryotic tRNA Ala (for example, yeast tRNA Ala ) The m 1 I37 is synthesized from A37 via I37 . Holley et al., 1965;Grosjean et al., 1996;Brulé et al., 2004 yW37 derivatives For information on the biosynthetic pathways of yW37 derivatives, please see these references, Noma et al., 2010;de Crécy-Lagard et al., 2010 Biosynthesis of yW37 derivatives starts with the m 1 G37 modification by Trm5 in both eukaryotes and archaea Some archaeal biosynthetic pathways are predicted from genomic information In some intermediate steps, methylation(s) by Tyw3 (yeast) or Taw3 and Trm5 homologs (archaea) and a radical SAM reaction are involved The names, Trm12 and TrmM, were previously allocated to eukaryotic and archaeal methyltransferases, respectively in yW37 biosynthetic pathways Therefore, some genome sequence projects used these names
Half-life of tRNA Val is shortened in the yeast trm8 and trm4 double knockout strain Gene disruption of Trm8 homolog in Colletotrichum lagenarium causes the loss of infectious ability In the case of T. thermophilus, m 7 G46 modification is one of the key factors in network of modified nucleotides and tRNA modification enzymes and is essential for growth at high temperatures. Please see main text Alexandrov et al., 2002Alexandrov et al., , 2005Alexandrov et al., , 2006De Bie et al., 2003;Okamoto et al., 2004;Cartlidge et al., 2005;Takano et al., 2006;Zegers et al., 2006;Matsumoto et al., 2007;Chernyakov et al., 2008;Leulliot et al., 2008;Tomikawa et al., 2008Tomikawa et al., , 2010

MULTIPLE REGULATION OF tRNA MODIFICATION PATHWAYS AND IMPORTANCE OF THE AVAILABILITY OF METHYL DONORS
In living cells, more than 50% of the high energy compounds such as ATP, that are produced by respiration are consumed by protein synthesis. Furthermore, the most important metabolic pathway of amino acids is protein synthesis. The metabolic pathways of energy and amino acids are closely linked. Studies on the pathways of tRNA modification have revealed that the RNA modification systems are located downstream of the pathways of energy and amino acid metabolism and that they are regulated at multiple steps (Herbig et al., 2002;Iwata-Reuyl, 2003;Ikeuchi et al., 2008Ikeuchi et al., , 2010Shigi et al., 2008;Suzuki and Miyauchi, 2010;Phillips et al., 2012;Laxman et al., 2013;Miyauchi et al., 2013;Perrochia et al., 2013 Figure 3 and Table 1). Thus, depletion of a certain compound (for example, one of the amino acids) or disruption of a metabolic pathway can result in incomplete modification of tRNA and thus an increased frequency of translational errors. The structures of identified modified nucleosides suggest that the majority of tRNA modifications require a methylation step(s) (Table 1 and Figure 1). The methyl-transfer reaction by majority of tRNA methyltransferases consumes S-adenosyl-L-methionine (AdoMet) as the methyl-group donor. Thus, the depletion of AdoMet leads to multiple incomplete modifications in tRNA. The precursors of AdoMet are ATP and methionine. These facts seem to provide an answer for the question, "Why do living organisms use the methionine codon as the initiation codon for protein synthesis?" Under conditions where methionine is limited and the tRNA contains multiple incomplete modifications, to avoid increase of frequency of translational error, the methionine codon is selected the initiation codon of protein synthesis. Analogously, the fact that eubacterial methionyl-initiator tRNA Met is formylated and formylation is the transfer of one carbon atom suggests that the supply of sources of single carbon atoms is very important for efficient and accurate protein synthesis in bacteria.

STRUCTURES OF tRNA METHYLTRANSFERASES
Transfer RNA methyltransferases can be divided into two types on the basis of their methyl donor: one class uses AdoMet whereas the other utilizes 5, 10-methylenetetrahydrofolate ( Table 2). As mentioned above, the majority of tRNA methyltransferases are AdoMet-dependent. For information on the catalytic mechanisms of tRNA methyltransferases, (see Watanabe et al., 2005;Kuratani et al., 2008;Meyer et al., 2008;Osawa et al., 2009;Hou and Perona, 2010;Hamdane et al., 2012). Recently, a radical SAM enzyme was identified as a ribosomal RNA methyltransferase (Atta et al., 2010); radical SAM enzymes utilize a 4Fe-4S cluster to generate a reactive radical from AdoMet. No radical SAM enzymes that act as tRNA methyltransferases have been identified as yet. However, three types of radical SAM enzymes are involved in tRNA modifications (2-methylthiotransferases that generate ms 2 t 6 A derivatives, 2-methylthiotransferases that generate ms 2 i 6 A derivatives, and enzymes involved in the biosynthesis of yW37 derivatives) (Suzuki et al., 2007;Atta et al., 2010Atta et al., , 2012de Crécy-Lagard et al., 2010;Fujimori, 2013 and Table 1). Radical SAM tRNA methyltransferase(s) might be identified in the near future, because there are many methylated nucleosides, for which the responsible enzyme(s) have not yet been identified (Table 1).
AdoMet-dependent methyltransferases are classified by their catalytic domain (Schubert et al., 2003). Two different classes (classes I and IV) have been identified among the tRNA methyltransferases ( Table 2). Class I enzymes contain the Rossmann fold in the catalytic domain (Figure 4A), whereas class IV enzymes have the topological-knot structure ( Figure 4B). Class IV enzymes were predicted initially by bioinformatics studies to be members of the SpoU-TrmD (SPOUT) superfamily (Anantharaman et al., 2002). Subsequently, crystallographic studies (Table 2) revealed that these enzymes have a topological knot structure. YibK was predicted initially to be an RNA methyltransferase of unknown function (Gustafsson et al., 1996). Determination of the crystal structure revealed the presence of the topological-knot structure in the catalytic domain of YibK (Lim et al., 2003). Later, YibK was shown to function as tRNA (Cm34/cmnm 5 Um34) methyltransferase and was renamed TrmL (Benítez-Páez et al., 2010;Liu et al., 2013). At almost the same    (Gustafsson et al., 1996;Anantharaman et al., 2002;Purta et al., 2006;Roovers et al., 2008a;Phizicky and Hopper, 2010;Tkaczuk, 2010). Detailed insight into catalytic mechanisms of tRNA methyltransferases is only available in a few cases: see these references Kuratani et al., 2008;Meyer et al., 2008;Osawa et al., 2009;Hou and Perona, 2010;Hamdane et al., 2012).
time, three groups independently reported the crystal structures of TrmD proteins and revealed that TrmD proteins also contain the topological-knot structure (Ahn et al., 2003;Elkins et al., 2003;Liu et al., 2003). In 1997, SpoU was found to have tRNA (Gm18) 2 -O-methyltransferase activity and was renamed as TrmH (Persson et al., 1997). We solved the crystal structure of TrmH in 2004 and confirmed that it is a class IV enzyme with the topological-knot structure (Nureki et al., 2004 and Figure 4C). These studies established the structural foundation of SPOUT enzymes (Anantharaman et al., 2002;Tkaczuk et al., 2007), which can be identified on the basis of the topological-knot structure.
To date, several tRNA methyltransferases have been identified as members of the SPOUT superfamily on the basis of crystal structures (Kuratani et al., 2008;Chen and Yuan, 2010;Chatterjee et al., 2012;Wurm et al., 2012;Shao et al., 2013) or structures predicted from amino acid sequences and conserved motifs (Renalier et al., 2005;Purta et al., 2006;Tkaczuk et al., 2007;Kempenaers et al., 2010, and Figure 4B). Furthermore, the SPOUT superfamily is expanding beyond the SpoU and TrmD families: novel enzymes such as an archaeal Trm10 homolog (Kempenaers et al., 2010) and TrmY (Chen and Yuan, 2010;Chatterjee et al., 2012;Wurm et al., 2012) have been identified. These enzymes cannot be simply classified into the SpoU or TrmD families. Therefore, it might be necessary to reclassify the enzymes of the SPOUT superfamily on the basis of their structure, the methylated nucleosides produced, and their reaction mechanisms. The number of identified class I methyltransferases has also increased. Crystal structures of class I enzymes have been reported, as shown in Table 2; however, for many of the enzymes, structures have been predicted from their amino acid sequences and conserved motifs. The difficulty with crystallographic studies is that the eukaryotic and archaeal enzymes often require other subunit(s) to regulate (or stabilize) their activities (Anderson et al., 1998;Alexandrov et al., 2002Alexandrov et al., , 2005Purushothaman et al., 2005;Mazauric et al., 2010;Liger et al., 2011;Noma et al., 2011, and Table 1). Only a few structural studies of the multisubunit complexes have been performed, namely Trm8-Trm82 (Leulliot et al., 2008), and the Fibrillalin, Nop5 and L7Ae complex (Ye et al., 2009;Lin et al., 2011). In addition, structures for the tRNA bound-form of Trm5 (Goto-Ito et al., 2009) and T-armlike RNA bound-form of TrmA (Alian et al., 2008) have been reported. Furthermore, several eukaryotic tRNA methyltransferases are fused with other functional domains and are involved in other processes such as DNA repair (Choudhury et al., 2007a,b;Shimada et al., 2009;Fu et al., 2010;Songe-Møller et al., 2010;D'Silva et al., 2011;Leihne et al., 2011;Noma et al., 2011;van den Born et al., 2011;Pastore et al., 2012). Although the crystal structures of the RNA recognition motif and AlkB domains of ALKB8H, which also contains a methyltransferase domain, have been reported (Pastore et al., 2012), there is no entire crystal structure of a eukaryotic multidomain tRNA methyltransferase. To understand the reaction mechanisms, substrate specificity, subunit (domain) interactions, and regulation of activity of these enzymes, structural studies are necessary.
As studies on eukaryotic enzymes have progressed, the number of complex enzymes identified has increased. For example, mammalian enzymes often have additional domains, regulatory subunits and/or paralogs. For information on the identification and prediction of human tRNA methyltransferases, see this review (Towns and Begley, 2012).

TRANSFER RNA RECOGNITION BY tRNA METHYLTRANSFERASES
Transfer RNA methyltransferases strictly modify a specific nucleoside at a specific position in a tRNA. Within the field of nucleic acid-related enzymes, a common question is "How does the enzyme recognize a specific substrate and act at a specific position?" Consequently, the substrate specificities of tRNA methyltransferases have been studied by measuring activities in crude cell extracts, microinjecting labeled tRNA, biochemical studies with purified enzymes, crystallographic studies, and analyses of tRNA from disruptant strains.
In general, tRNA methyltransferases recognize the local structure around the target site in the tRNA, including tertiary structural elements such as stem-loop structure(s). TrmA from E. coli recognizes U54 in the ribose-phosphate backbone of the T-arm (Gu and Santi, 1991;Alian et al., 2008). Aquifex aeolicus TrmB requires the five nucleotides AGG * UC sandwiched between two stem-loop structures (the asterisk corresponds to the methylation site, G46) (Okamoto et al., 2004). TrmFO recognizes the G53-C61 base pair and U54U55C56 sequence in the T-arm (Yamagami et al., 2012). TrmD recognizes the purine36G37 sequence in the anticodon-arm-like microhelix (Brulé et al., 2004;Takeda et al., 2006). In some cases, tertiary interactions are required. For example, crystallographic studies of the complex between Trm5 and tRNA revealed that the enzyme requires interaction between the D-and T-loop of the tRNA (Goto-Ito et al., 2009), which is consistent with the results of biochemical studies with the purified enzyme (Christian et al., 2004;Christian and Hou, 2007).
The target site for methylation is often embedded in the L-shaped tRNA structure. Consequently, in many (or almost all) cases, recognition of tRNA by tRNA methyltransferases seems to involve multiple steps (initial binding and induced fit processes).
Although it is very difficult to prepare intermediate complexes, we recently analyzed the initial binding and changes in structure of TrmH by stopped-flow presteady-state kinetic analysis (Ochi et al., 2010(Ochi et al., , 2013. TrmH binds to tRNA within 10 ms in the initial binding process, in which substrate and non-substrate (methylated) tRNAs are not distinguished. Methylated tRNA is excluded from the complex subsequently due to steric hindrance between the methyl groups in the tRNA and AdoMet before the induced-fit process occurs. The advantage of this mechanism is that methylated tRNA does not severely inhibit the methyl-transfer reaction as a competitive inhibitor. Subsequently, in the induced-fit process, which takes more than 50 ms, G18 is recognized and ribose introduced into the catalytic pocket. During the induced-fit process, movement of Trp126 in motif 2 is observed (Ochi et al., 2013 and Figure 4C).
Several tRNA methyltransferases act on multiple sites in tRNA. For example, archaeal TrmI acts on both A57 and A58 Guelorget et al., 2010). Similarly, Aquifex aeolicus Trm1 acts on both G26 and G27 (Awai et al., 2009). On the basis of biochemical studies, we determined that this eubacterial Trm1 recognizes the methylation sites (G26 and G27) from the T-arm (Awai et al., 2009(Awai et al., , 2011 whereas archaeal Trm1 recognizes G26 from the D-stem and variable region (Constantinesco et al., 1999b). These Trm1 proteins share high sequence homology (Awai et al., 2009); however, comparison of the crystal structures revealed that the distribution of positive charges on the enzyme surface differs between archaeal (Ihsanawati et al., 2008) and eubacterial (Awai et al., 2011) Trm1. Thus, these studies show how difficult it is to predict target sites on the basis of amino acid sequences. Furthermore, in some cases, other subunits regulate the site specificity. For example, the methylation site recognized by Trm7 is determined by its partner subunit (Guy et al., 2012) and the site specificity of archaeal Trm4 changes in the presence of archease . Moreover, the m 5 C modifications in eukaryotic tRNA are regulated by the presence of an intron in the precursor tRNA (Motorin and Grosjean, 1999;Brzezicha et al., 2006;Auxilien et al., 2012). In addition, some 2 -O-methylated nucleosides in archaeal tRNA are introduced by the aFib, Nop5p and L7Ae complex with the BoxC/D guide RNA system (Clouetd'Orval et al., 2001Bortolin et al., 2003;Singh et al., 2004;Renalier et al., 2005;Ye et al., 2009;Joardar et al., 2011;Lin et al., 2011). In some cases, an intron in the precursor tRNA acts as the guide RNA (Clouet-d'Orval et al., 2001Bortolin et al., 2003;Singh et al., 2004). This system is useful in minimizing the size of the genome. In the future, it is possible that considerable numbers of 2 -O-methylated modifications in archaeal tRNA might be identified as products of this system.

REGULATION OF THE DEGRADATION AND LOCALIZATION OF tRNA BY METHYLATED NUCLEOSIDES
As shown in Table 1, modifications of the anticodon loop (positions 32-38) are involved directly in protein synthesis whereas other modifications affect the structure of the tRNA. Consequently, for a long time, it was thought that modifications outside the anticodon loop acted to stabilize tRNA structure and regulate the half-life of tRNAs. Indeed, we observed in the thermophilic eubacterium Thermus thermophiles that hypomodification at multiple sites in tRNA owing to disruption of one of the modification enzymes promotes the degradation of tRNA Phe and tRNA Lys at high temperatures (Tomikawa et al., 2010).
In the case of eukaryotes, tRNA methylations work coordinately as stabilizing factors and markers of maturation, and the degree of modification changes in response to various stresses. Hypomodified tRNAs are degraded aggressively. For example, in the Saccharomyces cerevisiae trm4 (synthesizes m 5 C at multiple sites) and trm8 (produces m 7 G46) double knock-out strain, the half-life of tRNA Val is shortened and the strain shows a growth defect (Alexandrov et al., 2006). Therefore, tRNA modifications stabilize tRNA structure coordinately and systems to degrade hypomodified tRNAs exist in eukaryotic cells (Alexandrov et al., 2006;Chernyakov et al., 2008;Phizicky and Hopper, 2010;D'Silva et al., 2011;Dewe et al., 2012). Furthermore, in S. cerevisiae, the m 1 A58 modification by the Trm6-Trm61 complex regulates both the degradation of initiator tRNA Met and its transport from the nucleus to the cytoplasm (Anderson et al., 1998(Anderson et al., , 2000Kadaba et al., 2004). The m 1 A58 modification functions a marker of maturation and absence of modification leads to degradation of initiator tRNA Met during transport. Thus, m 1 A58 is part of the RNA quality control system. Moreover, in the case of S. cerevisiae, splicing is performed in the cytoplasm (Takano et al., 2005) and precursor tRNAs are matured during repeated-transports between the nucleus and cytoplasm (Ohira and Suzuki, 2011). Therefore, some tRNA modifications might act as the markers of maturation at halfway checkpoints. In Leishmania tarentolae, a proportion of tRNA Glu and tRNA Gln is transported from the cytoplasm to the mitochondria (Kaneko et al., 2003). In the cytoplasmic tRNA, U34 is modified to mcm 5 s 2 U34, whereas in the mitochondrial tRNA it is modified to mcm 5 Um34. These results suggest that the s 2 U34 modification may suppress transport from the cytoplasm to mitochondria. Given that both the s 2 U and Um modifications shift the equilibrium of ribose puckering to the C3 -endo form (Kawai et al., 1992), these modifications have a nearly equivalent stabilizing effect on the codon-anticodon interaction. The 5-methylcarboxymethyl (mcm) group restricts wobble base pairing (Takai and Yokoyama, 2003). Taken together, these findings suggest that a substantial number of methylated nucleosides contribute to RNA quality control systems and/or the regulation of tRNA localization, even though they were considered previously to have simply a structural role.

ADAPTATION OF PROTEIN SYNTHESIS TO ENVIRONMENTAL CHANGE THROUGH A NETWORK BETWEEN MODIFIED NUCLEOSIDES AND tRNA MODIFICATION ENZYMES tRNA MODIFICATIONS IN T. THERMOPHILUS
Thermus thermophilus provides an example of a living organism that utilizes changes in the structural rigidity (flexibility) of tRNA through multiple nucleoside modifications to adapt protein synthesis to environmental changes. Thermus thermophilus is an extreme thermophilic eubacterium found in hot springs and can grow at a wide range of temperatures (50∼83 • C ). Under natural conditions, the temperature of hot springs can be changed dramatically by several factors, for instance the overflow of hot spring water, snow falling, and the influx of river water. Thermus thermophilus can synthesize proteins in response to these temperature changes. Three distinct modifications (Gm18, m 5 s 2 U54, and m 1 A58) are found in T. thermophilus tRNA and the combination of these modifications increases the melting temperature of tRNA by near 10 • C as compared with that of the unmodified transcript (Watanabe et al., 1976;Horie et al., 1985;Shigi et al., 2006;Tomikawa et al., 2010). Although these modifications are very important as structural factors in tRNA, they do not have an effect on translational fidelity below 65 • C and the level of modification is very low in tRNA from cells cultured at 50 • C ( Figure 5A). This change in the extent of modification reflects the adaptation of protein synthesis to temperature change (Yokoyama et al., 1987). Transfer RNA Phe from cells cultured at 80 • C efficiently synthesizes poly(U) at high temperatures (above 65 • C). In contrast, tRNA Phe from cells cultured at 50 • C, in which the levels of the three modifications are low, works efficiently at low temperatures ( Figure 5B). Thus, the levels of three modified nucleosides, Gm18, m 5 s 2 U54, and m 1 A58, in tRNA control the elongation of translation via the flexibility of the tRNA. These findings were The m 1 A58 modification accelerates the thio-transfer reaction by the sulfur atom exchange complex that is required for the formation of m 5 s 2 U54. Therefore, at high temperatures, m 7 G46, m 5 U54, and m 1 A58 coordinately promote the formation of m 5 s 2 U54 and increases tRNA stability. In contrast, at low temperatures below 65 • C, the ψ55 modification increases rigidity within the local structure of the tRNA as described in the main text. This network provides a mechanism by which extreme thermophilic eubacteria adapt to temperature changes. The network regulates the order of modifications in tRNA. This figure summarizes the experimental data in these publications Yokoyama et al. (1987), Shigi et al. (2006), Tomikawa et al. (2010), Ishida et al. (2011), Yamagami et al. (2012 reported in 1987 (Yokoyama et al., 1987). However, at the beginning of the twenty-first century, the mechanisms of regulation of these modifications remained unknown.

SWITCHING OF NETWORK BETWEEN MODIFIED NUCLEOSIDES AND tRNA MODIFICATION ENZYMES
Initially, we assumed that transcriptional and/or translational regulation of the tRNA modification enzymes was involved in the regulation of the three modifications. However, unexpectedly, we have observed that the phenomenon can be simply explained by the RNA recognition mechanisms of the tRNA modification enzymes (Shigi et al., 2002;Tomikawa et al., 2010;Ishida et al., 2011;Yamagami et al., 2012). Several common modifications (for example, m 7 G46 and ψ55) are found in T. thermophilus tRNA in addition to Gm18, m 5 s 2 U54, and m 1 A58. When the genes for the modification enzymes for m 7 G46 and ψ55 (trmB and truB, respectively) were disrupted individually, the levels of Gm18, m 5 s 2 U54 and m 1 A58 in tRNA were changed dramatically (Tomikawa et al., 2010;Ishida et al., 2011). Thus, modified nucleosides and tRNA modification enzymes form a network, and this network regulates the extent of modifications on the basis of temperature (Figures 5C,D).
At high temperatures (above 65 • C), m 7 G46 functions as a marker of precursor tRNA and increases the reaction rates of other modification enzymes. In contrast, at low temperatures, ψ55 confers local structural rigidity and slows down the rate of formation of other modifications around ψ55 (that is, Gm18, m 5 s 2 U54, and m 1 A58). This inhibitory effect weakens as the temperature increases and is not observed above 65 • C. Thus, the m 7 G46 and ψ55 modifications work as an accelerator and a brake in the network, respectively. The advantage of this mechanism is that the network does not include any transcriptional or translational regulatory steps: protein synthesis is not necessary. Thus, the response of the network to environmental changes is very rapid. This is a typical strategy in eubacteria, where genome size is limited. Similar networks between modified nucleosides and tRNA modification enzymes have also been reported in mesophiles. For example, ms 2 i 6 A37 modification in E. coli tRNA is required for 2 -O-methylation by TrmL (Benítez-Páez et al., 2010), and the Cm32 and Gm34 modifications in S. cerevisiae tRNA Phe are required for the formation of yW37 from m 1 G37 (Guy et al., 2012). However, the network in T. thermophilus is distinct because the modifications are almost all in the three-dimensional core of the tRNA and the network responds to environmental changes.

GENETIC DISEASE AND tRNA METHYLATION
Modifications of tRNA regulate protein synthesis. Consequently, if a disruption of tRNA modification is not lethal, it can directly cause a genetic disease. In fact, there are several reports concerning the relationship between genetic disease and tRNA modification (Yasukawa et al., 2001;Suzuki et al., 2002Suzuki et al., , 2011bFreude et al., 2004;Kirino et al., 2005;Umeda et al., 2005;Wei et al., 2011;Towns and Begley, 2012;Igoillo-Esteve et al., 2013). In particular, the number of reports of a link between diabetes and tRNA modification are increasing, which suggests that an increase in the frequency of translation errors has an effect on energy metabolism. The severe disruption of energy metabolism often damages muscle and neuronal cells, which consume large amounts of energy. This perspective enables mitochondrial diseases that are caused by a problem with mitochondrial tRNA modification to be understood (Yasukawa et al., 2001;Suzuki et al., 2002Suzuki et al., , 2011bKirino et al., 2005;Umeda et al., 2005). Furthermore, several tRNA methyltransferases are fused to DNA repair enzymes, which means that these enzymes are related directly to DNA repair and carcinogenesis (Choudhury et al., 2007a,b;Shimada et al., 2009). Moreover, abnormal tRNA modifications have been also reported in cancers (Kuchino and Borek, 1978;Kuchino et al., 1981;Shindo-Okada et al., 1981). These might be caused by the rearrangement of chromosomes in cancer cells.

INFECTION, IMMUNITY, AND tRNA METHYLATIONS-tRNA THERAPY
Among the tRNA modification enzymes, tRNA guanine transglycosidase (Tgt), which is required for the production of Q34, and tRNA ψ55 synthase (TruB), which generates ψ55, are essential factors for infection by Shigella flexneri (Durand et al., 1994) and Pseudomonas aeruginosa (Saga et al., 1997), respectively. Similarly, we also found that tRNA (m 7 G46) methyltransferase is essential for infection by Colletotrichum lagenarium, an infectious fungus . Furthermore, tRNAs that contain mcm 5 U modifications are the target of Kluyveromyces lactis gamma-toxin (Lu et al., 2005) and Pichia acaciae killer toxin (Klassen et al., 2008). Moreover, given that retroviruses utilize host tRNA as the primer for reverse transcription, tRNA methylation and methyltransferases are involved in both reverse transcription and the packaging of virus particles. For example, human immunodeficiency virus (HIV; AIDS virus) utilizes the m 1 A58 modification in tRNA Lys 3 as the terminator of reverse transcription (see reviews, Marquet, 1998;Saadatmand and Kleiman, 2012;Sleiman et al., 2012). Consequently, the regulation of tRNA modification and modification enzymes might be a powerful tool to control infectious organisms.
When an exogenous single-stranded RNA such as Haemophilus influenzae tRNA is present in humans, Tolllike receptor 7 (TLR7) forms a dimer structure and then activates the immune response systems (Figure 6). However, endogenous or E. coli tRNA does not stimulate TLR7. The mechanism of differentiation was clarified recently by two groups, who found that the Gm18 modification in E. coli tRNA suppresses immunostimulation via TLR7 (Gehrig et al., 2012;Jöckel et al., 2012). Thus, enterobacteria exploit the Gm18 modification in tRNA to avoid the host immune system. Furthermore, given that Gm18-modified tRNA acts as an antagonist of TLR7 (Jöckel et al., 2012), Gm18-modified tRNA might be an effective anti-inflammatory drug.

EVOLUTION OF MODIFICATIONS IN tRNA
Finally, it is worthwhile discussing the evolution of modifications in tRNA. During the early period of chemical evolution (see reviews Cermakian and Cedergren, 1998;Joyce and Orgel, 2006), inosine could be used as a basic component of RNA, because it can be synthesized from adenosine non-enzymatically. Inosine seems to have been excluded after the appearance of genes because it changes the genetic information during the replication process. Simple methylated nucleosides such as m 1 G became essential when the reading frame of protein synthesis was separated into three-nucleotide units (Björk et al., 1989. Thus, several methylated nucleosides seem to have appeared during the chemical evolution period (Cermakian and Cedergren, 1998). After the appearance of the reading frame, the importance of the availability of methyl groups increased and it seems that the methionine codon was selected as the translation initiation codon.
It appears that complicated enzymes were not formed during the period of chemical evolution (Joyce and Orgel, 2006). The early enzymes might have been oligopeptides and might have included metals as the catalytic center, as is the case for deaminases (Carter, 1998;Schaub and Keller, 2002). It is possible that the codons were not fixed strictly as is observed in the universal code (Jukes, 1973;Cedergren et al., 1986;Osawa et al., 1992). However, it is likely that the most basic catalytic core of tRNA methyltransferases was established when cell-like organisms began to exchange their components and genes because the basic structure of tRNA methyltransferases is shared by all living organisms found today (Figure 4 and Table 2). The structures of methyltransferases (Schubert et al., 2003) suggest that RNA methyltransferases, which were required for protein synthesis, evolved to yield DNA and protein methyltransferases many times during the evolution of life. The mechanisms to generate the complicated modified nucleotides that regulate the wobble base pair seem to have arisen after the origination of living organisms because they show considerable diversity and involve multistep reactions ( Table 1).
The temperature of primordial Earth was higher than that of the Earth at present. Consequently, several nucleoside modifications in tRNA and rRNA would be necessary to stabilize the structure of the RNA (Motorin and Helm, 2010). However, it is likely that the network between modified nucleosides and tRNA modification enzymes that is observed in extreme thermophiles ( Figure 5D and section Adaptation of Protein Synthesis FIGURE 6 | Immune response and tRNA methylation. Transfer RNA from H. influenzae, a respiratory infectious bacterium, induces the dimer formation by Toll like receptor-7 (TLR7) and then the immune response is stimulated via binding of the proteins, MyD88, IRAK1, and IRAK4. However, human and E. coli tRNAs do not stimulate TLR7 because these tRNA contains the Gm18 modification. The E. coli trmH gene disruptant strain does not show any obvious phenotype under laboratory culture conditions (Persson et al., 1997). The Gm18 modification and TrmH are required for survival of E. coli in the animal gut.
to Environmental Change Through a Network Between Modified Nucleosides and tRNA Modification Enzymes) was established after the cooling of the Earth because it responds to low temperatures (Ishida et al., 2011). Obviously, the functions of modified nucleotides with respect to the RNA quality control system and regulation of cellular localization were acquired after the appearance of eukaryotes (see section Regulation of the Degradation and Localization of tRNA by Methylated Nucleosides).
Transfer RNA modifications are still evolving. The most powerful driving force is the existence of infectious organisms (see section Infection, Immunity, and tRNA Methylations-tRNA Therapy). Hosts need to distinguish endogenous RNA from exogenous RNA to prevent infection and infectious organisms need to avoid the host defense system to survive. Consequently, tRNA modifications and modification enzymes are still subject to evolution even today.