Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA

Abstract

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted. Therefore, thermophiles must possess strategies to stabilize tRNA structures. To this end, several thermophile-specific modified nucleosides in tRNA have been identified. Other factors such as RNA-binding proteins and polyamines contribute to the stability of tRNA at high temperatures. Thermus thermophilus, which is an extreme-thermophilic eubacterium, can adapt its protein synthesis system in response to temperature changes via the network of modified nucleosides in tRNA and tRNA modification enzymes. Notably, tRNA modification enzymes from thermophiles are very stable. Therefore, they have been utilized for biochemical and structural studies. In the future, thermostable tRNA modification enzymes may be useful as biotechnology tools and may be utilized for medical science.

1. Introduction

Transfer RNA is an adaptor molecule required for the conversion of genetic information encoded by nucleic acids into amino acid sequences of proteins [1,2]. Figure 1A shows typically conserved nucleosides in a tRNA molecule, which is represented as a cloverleaf structure (herein, the nucleotide positions in tRNA are numbered, according to Sprinzl et al. [3]). These conserved nucleotides are important for tRNA folding and for stabilization of the L-shaped tRNA structure (Figure 1B) [4,5,6]. In addition to the standard nucleosides, numerous modified nucleosides in tRNA (for structures, see the MODOMICS and tRNAmodviz databases: http://modomics.genesilico.pl/; http://genesilico.pl/trnamodviz [7]) have been discovered in both thermophilic and mesophilic tRNAs [7,8] (see Supplementary Table S1 for abbreviations of modified nucleosides).

Figure 1.

The structure of tRNA. (A) Representation of secondary structure of tRNA in a cloverleaf structure. This figure shows tRNA with a short variable region. Conserved nucleosides are shown with position numbers. Abbreviations: R, purine. Y, pyrimidine. (B) The L-shaped structure of Saccharomyces cerevisiae tRNA^Phe. The colors of nucleosides correspond to those in (A).

A comprehensive review of the modified nucleosides in tRNA from thermophiles and their positions, distribution, predicted (or confirmed) tRNA modification enzymes and structural effects (Table 1) [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264], which suggests that the majority of modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles. The functions of modified nucleosides in tRNAs have been gradually elucidated by biochemical and structural studies, physicochemical measurements, and analyses of gene disruption strains. The modified nucleosides primarily function in protein synthesis (e.g., stabilization of tRNA structure [88,265,266,267], correct folding of tRNA [88,265,266,267], reinforcement, restriction, and/or alteration of codon-anticodon interaction [108,109,114,115,116,117,120,124,268,269,270], recognition by aminoacyl-tRNA synthetases [109,116,117,271], recognition by translation factors [272], and prevention of the frameshift error [122,123,157,158] among others). In short, living organisms cannot synthesize proteins correctly or efficiently without modifications in tRNA.

Table 1.

Modified nucleosides in tRNA from thermophiles.

Modified Nucleoside and Position	Distrib.	Modification Enzyme	Predicted Functions and Additional Information	References
Am6	A	Unknown	Stabilization of aminoacyl-stem Enzymatic activity for Am6 formation has been detected in the cell extract of Pyrococcus furiosus	[9]
m2G6	B/A	TrmN/Trm14	Stabilization of aminoacyl-stem	[10,11,12,13,14,15]
U8	A	CDAT8	Increasing G-C content in tRNA genes In Methanopyrus kandleri, U8 in several tRNA is produced from C8 by the deamination [16] In Methanopyrus kandleri, numerous nucleosides in RNA may be 2-O-methylated (see main text) [17]	[16,17]
s4U8	B/A	ThiI + IscS/ThiI	UV resistance in E. coli and Salmonella typhimurium (see main text) Stabilization of D-arm structure in E. coli (see main text)	[10,11,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35]
s4U8 and s4U9	A	ThiI + α?	UV resistance Stabilization of D-arm structure (see main text) Sulfur-containing modifications in tRNA are reviewed in Reference [35].	[36]
m1A9	A	Archaeal Trm10	Stabilization of the D-arm structure Prevention of formation of a Watson–Crick base pair Correct folding of the D-arm region	[37,38]
m1G9 and m1A9	A	archaeal Trm10	Stabilization of D-arm structure Prevention of formation of a Watson–Crick base pair Correct folding of D-arm region Thermococcus kodakarensis Trm10 forms m1G9 and m1A9, whereas Sulfolobus acidocaldarius Trm10 forms only m1A9	[37,39]
(m2G10 and) m22G10	A	archaeal Trm11 (Trm-G10; Trm-m22G10 enzyme)	Prevention of formation of a Watson-Crick base pair Correct folding of tRNA in Pyroccocus furiosus Correct folding of the D-arm region	[40,41,42,43]
Ψ13	B/A	TruD/TruD or archaeal Pus7	Stabilization of D-stem structure Archaeal Pus7 generally catalyzes formation of Ψ35 in tRNATyr, but Sulfolobus solfaraticus Pus7 has weak Ψ13 formation activity [46]	[23,44,45,46]
G+13	A	ArcTGT + ArcS?	Stabilization of the D-arm structure Thermoplasma acidophilum tRNALeu exceptionally possesses a G+13 modification and T. acidophilum ArcTGT acts on positions 13 and 15 in this tRNA [47]	[36,47]
G+15	A	ArcTGT + ArcS or QueF-like protein	Stabilization of interaction between the D-arm and the variable region Several archaea possess a split-type ArcTGT [60,61] Several species in Crenarchaeota possess a QueF-like protein instead of ArcS [60,62,63] G+ is not found in nucleosides from a Stetteria hydrogenophila tRNA mixture [56]	[25,36,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63]
D17	B	Dus family protein?	Maintenance of D-loop flexibility D17 and D20 modifications have been reported in Geobacillus stearothermophilus tRNA. However, D17 and D20 are formed by DusB and DusA, respectively, in Escherichia coli [65,66] and the G. stearothermophilus genome possesses only one dus-like gene. This is also observed in Bacillus subtilis, which is a mesophilic eubacterium.	[18,19,64,65,66]
Gm18	B	TrmH	Stabilization of the D-arm and the T-arm interaction. TrmH from thermophiles possess relative broad substrate tRNA specificities as compared with TrmH from E. coli. The substrate tRNA specificities of TrmH enzymes differ among thermophiles. TrmH from Thermus thermophilus can methylate all tRNA species.	[10,11,20,21,23,24,30,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81]
D20	B	Dus family protein	Stabilization of local structure of D-loop in E. coli? In A. aeolicus, the nucleosides at positions 20 and 20a in tRNACys are D20 and U20a, respectively. Therefore, Dus from A. aeolicus may act only on U20 in tRNA.	[24,33,65,66,82]
D20 and D20a	B	DusA	Stabilization of local structure of the D-loop The melting temperature of a tRNA mixture from the E. coli dusA gene disruptant strain is lower than that from the wild-type strain [33]. Therefore, D20 and D20a modifications may contribute to stabilize local structure of the D-loop. Thermus thermophilus Dus was recently confirmed as a member of the DusA family [65,66,84,85].	[21,22,23,33,65,66,67,83,84,85]
m1A22	B	TrmK	Prevention of formation of a Watson–Crick base pair	[18,20,86]
Ψ22	A	Unknown	The Ψ13-Ψ22 base pair may stabilize D-arm structure [88]	[87,88]
m2G26 and m22G26	A	Trm1	Stabilization of three-dimensional core structure Correct folding of tRNA Recently, it has been reported that m22G26 modification is required for correct folding of precursor tRNASer from Schizosaccharomyces pombe [94]. Therefore, a similar phenomenon may take place in thermophiles.	[9,25,44,89,90,91,92,93,94]
m2G26, m22G26, m2G27 and m22G27	B	Trm1	Stabilization of three-dimensional core structure in A. aeolicus. In the case of m2G27 and m22G27, stabilization of aminoacyl-stem	[24,95]
m22Gm26	A	Trm1 + unknown MT	Stabilization of three-dimensional core structure The presence of m22Gm has been confirmed in nucleosides of a tRNA mixture from several thermophilic archaea [56,97,98,99,100]. Although the nucleoside at position 26 in S. acidocaldarius tRNAMeti was originally reported as an unidentified G modification [44], it was recently described as m22Gm [96]. The MT for 2’-O-methylation is unknown.	[44,96]
Cm32	A	archaeal TrmJ	Stabilization of anticodon-loop	[96]
Cm32 and Nm32	B	TrmJ	Stabilization of anticodon-loop TrmJ from E. coli does not recognize the base at position 32 [96,102]. Um32 and Am32 have not been reported in tRNAs from thermophilic eubacteria.	[96,101,102]
I34	B	TadA	Alteration of codon–anticodon interaction A-to-I editing in tRNA is reviewed in Reference [107]	[103,104,105,106,107]
k2C34	B	TilS	Alteration of codon–anticodon interaction (E. coli and B. subtilis) Change of recognition by aminoacyl-tRNA synthetase (E. coli and B. subtilis) Decoding of AUA codons by k2C34 and agm2C34 modifications is reviewed in References [114,115].	[108,109,110,111,112,113]
agm2C34	A	TiaS	Alteration of codon–anticodon interaction (Arhaeoglobus fulgidus and Haloarcula marismourtui) Change of recognition by aminoacyl-tRNA synthetase (Arhaeoglobus fulgidus and Haloarcula marismourtui) Decoding of AUA codons by k2C34 and agm2C34 modifications is reviewed in References [114,115].	[114,115,116,117,118,119,120]
xm5U34 derivatives	B/A	MnmE + MnmG + MnmC (for mnm5U34 in eubacteria)/Elp3? + α (for cm5U34 in archaea) IscS + TusA + TusBCD + TusE + mnmA (for 2-thiolation in E. coli) or YrvO + mnmA (for 2-thiolation in B. subtilis) SAMP2 + UbaA + NcsA (for 2-thiolation in M. maripuludis)	Reinforcement of codon–anticodon interaction (E. coli and other mesophiles) Restriction of wobble base pairing (E. coli and other mesophiles) Prevention of frameshift errors (E. coli and other mesophiles) Biosynthesis pathways of xm5U34 derivatives are not completely clarified. Although the information on xm5U34 derivatives in tRNA from thermophiles is limited, the biosynthesis pathways may be common with those from mesophiles. For the functions and biosynthesis pathways for xm5U34 derivatives, see References [121,122,123,124,125,126,127,128,129,130,131,132,136,137,138,139,142]. For the thiolation of xm5s2U34 derivatives, see References [35,133,134,135]. Aquifex aeolicus exceptionally possesses a DUF752 protein, which is an MT for the xm5U34 modifications without an oxidase domain [136]. A mnm5U nucleoside has been found in modified nucleosides from unfractionated tRNA in several methane archaea [56]. Thermoplasma acidophilum tRNALeu possesses ncm5U34 [36]. Some thermophiles in Euryarchaea may have a cnm5U34 modification in tRNA [137]. The cm5U34 formation activity of Elp3 from Methanocaldococcus infernus has been reported [142]. Several related proteins for synthesis of xm5U34 derivatives from thermophiles have been used for structural studies [136,138,139,140,141].	[34,35,36,56,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142]
Cm34 and cmnm5Um34	B	TrmL	Reinforcement of codon–anticodon interaction (E. coli)	[18,143,144]
Gm34	B	Unknown	Reinforcement of codon–anticodon interaction (G. stearothermophilus)	[19]
Q34 derivatives	B	Tgt + QueA + QueG	Reinforcement of codon–anticodon interaction (E. coli) Prevention of frame-shift error (E. coli) Biosynthesis pathways and functions of Q derivatives are reviewed in References [152,153]. A crystal structure of QueA from T. maritima has been reported [151].	[20,122,145,146,147,148,149,150,151,152,153]
Cm34 and Um39 (or Cm39)	A	L7Ae + Nop5 + archaeal fibrillarin + Box C/D guide RNA (intron)	Reinforcement of codon–anticodon interaction Reinforcement of anticodon-arm In several archaea, an intron in precursor tRNATrp functions as a Box C/D guide RNA.	[9,154,155]
Ψ35	A	aPus7 and H/ACA guide RNA system	Reinforcement of codon–anticodon interaction	[46]
m1G37	B/A	TrmD/Trm5	Prevention of frame-shift error (E. coli and other mesophiles) Recognition by aminoacyl-tRNA synthetase (Saccharomyces cerevisiae)	[36,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171]
wyosine37 derivatives	A	Trm5 + Taw1 + Taw2 + Taw3	Reinforcement of codon–anticodon interaction Prevention of the frame-shift error In several archaea, m1G37 in tRNAPhe is further modified to wyosine derivatives. For the biogenesis pathway of wyosine derivatives, see References [181,182,183].	[172,173,174,175,176,177,178,179,180,181,182,183]
t6A37 derivatives	B/A	TsaB, TsaC (TsaC2), TsaD and TsaE/KEOPS complex: Kae1, Bud32, Cgi121 and Pcc1 + Sua5	Reinforcement of codon–anticodon interaction Prevention of frame-shift error Recognition by aminoacyl-tRNA synthetases The biogenesis pathway for t6A derivatives is reviewed in Reference [191]	[68,184,185,186,187,188,189,190,191]
i6A37 derivatives	B	MiaA + MiaB	Prevention of frame-shift error Reinforcement of codon–anticodon interaction Recognition by aminoacyl-tRNA synthetases i6A derivatives are reviewed in Reference [197]	[10,11,18,19,20,24,192,193,194,195,196,197]
m6A37	B	YfiC (TrmG?)		[64,198]
Ψ38, Ψ39 and Ψ40	B/A	TruA/Pus3	Prevention of frame-shift error (E. coli) Reinforcement of anticodon-arm	[10,11,18,19,20,23,87,199,200,201,202,203]
m7G46	B	TrmB	Stabilization of three-dimensional core In the case of T. thermophilus, m7G46 modification functions a key factor in a network between modified nucleosides in tRNA and tRNA modification enzymes (see main text) [11]	[10,11,19,67,204,205,206,207,208]
m5C48 and m5C49	A	archaeal Trm4	Stabilization of three-dimensional core	[9,209,210]
m7G49	A	Unknown		[36]
m5C51	A	Unknown	Stabilization of T-arm structure	[209]
m5C52	A	Unknown	Stabilization of T-arm structure	[209]
Ψ54 and Ψ55	A	Pus10	Stabilization of D-arm and T-arm interaction	[211,212,213,214]
m1Ψ54	A	Pus10 + TrmY	Stabilization of D-arm and T-arm interaction	[215,216,217]
m5U54 + m5s2U54	B/A	TrmFO + TtuA + TtuB + TtuC + TtuD + IscS/TrmA + TtuA? + TtuB? + α	Stabilization of D-arm and T-arm interaction (see main text) 2-Thiolation of m5s2U54 in tRNA is reviewed in Reference [239]	[10,11,21,22,23,24,67,97,98,134,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239]
Um54	A	Unknown	Stabilization of D-arm and T-arm interaction	[44]
Ψ55	B/A	TruB/Pus10 or archaeal Cbf5 + α	Stabilization of D-arm and T-arm interaction In the case of T. thermophilus, Ψ55 is required for low-temperature adaptation (see main text) [248].	[10,11,18,19,20,23,25,36,44,64,67,211,212,213,214,240,241,242,243,244,245,246,247,248]
Cm56	A	Trm56	Stabilization of D-arm and T-arm interaction	[9,25,36,44,48,89,249,250,251]
m2G57	A	Unknown		[44,252]
m1I57	A	archaeal TrmI + unknown deaminase	Stabilization of T-arm structure	[44,253,254]
m1A57 and m1A58	A	archaeal TrmI	Stabilization of T-arm structure	[44,255,256,257,258]
m1A58	B	TrmI	Stabilization of T-arm structure	[11,23,67,204,259,260,261,262,263,264]

This table shows the nucleosides that are modified in tRNA from thermophiles. Most modifications are common to those in tRNA from mesophiles. Several modifications include derivatives and they are summarized as the derivatives (e.g., xm⁵U₃₄ derivatives). In some cases, only modification enzymes from thermophiles have been reported. For example, although Q derivatives have not been confirmed in tRNA from T. maritima, the structure of QueA from T. maritima has been reported. In these cases, the modifications are listed here. The references for tRNA modifications and tRNA modification enzymes are mainly those for thermophiles. While there are many references for mesophiles, only representative references are cited. Where available, reviews of a modification and related proteins have been cited. Since modified nucleosides in tRNA from thermophilic eukaryotes have not been reported, modified nucleosides in eukaryotic tRNA have not been included here. The following modified nucleosides have been found in unfractionated tRNA from thermophiles. However, their positions and modified tRNA species are unknown: ac⁶A, hn⁶A, ms²hn⁶A, methyl-hn⁶A, m^{2, 7}Gm, s²Um, and ac⁴Cm [56,97,98,99,100]. Abbreviations are as follows: A, archaea, B, eubacteria, and MT, methyltransferase. The “?” mark indicates the potential function speculated from the structure of the modified nucleosides.

For some organisms, modifications in tRNA have not been confirmed, but the tRNA modification enzymes have been studied. For example, although no tRNA sequences from Thermotoga maritima have been reported, the properties of several tRNA modification enzymes of this hyper-thermophilic eubacterium have been documented and, thus, the nucleoside modifications are predicted. Although many of the functions and biosynthesis pathways of modified nucleosides in tRNA from thermophiles have not yet been investigated, most of them are considered to be basically common to those from mesophiles. However, thermophiles live in extreme environments (e.g., high temperature, anaerobic conditions, extreme pH, and high pressure). Therefore, it is possible that tRNA modifications observed in thermophiles may have novel functions. Furthermore, in some cases, the biosynthesis pathways of some modifications may differ between thermophiles and mesophiles. Moreover, in eukaryotes, tRNA modifications are related to higher biological processes such as cellular transport of tRNA [273,274,275,276,277,278], RNA quality control [274,279,280,281], infection [282,283,284,285,286], and the immune response [287,288,289,290]. As yet, modified nucleosides in tRNA from thermophilic eukaryotes have not been investigated, but it is possible that a relationship between modified nucleosides in tRNA and these biological phenomena may also be discovered in thermophilic eukaryotes.

In this review, we focus on the modified nucleosides and tRNA modification enzymes from thermophiles including the difficulties in sequencing the rigid and stable tRNAs from thermophiles. Since the tRNA modifications in moderate thermophiles are essentially similar to those in mesophiles, we describe them separately from extreme-thermophiles and hyper-thermophiles. We focus on the strategies for tRNA stabilization of extreme hyperthermophiles. Furthemrore, we describe the potential effects of these modifications during oxidative and other environmental stresses at high temperatures. Lastly, we describe biotechnological and therapeutic uses for tRNA modification enzymes. To avoid overlap with previous publications, we intentionally refer to representative articles and reviews of modified nucleosides in tRNA and tRNA modification enzymes from mesophiles (main text and Table 1) to aid understanding by the readers. For example, tRNA modifications in archaea including mesophiles have been extensively covered [48,87,291,292,293,294] and pseudouridine modifications and methylated nucleosides in tRNA are reviewed elsewhere [87,203,295,296]. Furthermore, the stability of nucleic acids at high temperatures has been reviewed [297]. Other useful publications are pointed out in the appropriate sections throughout the review.

2. Sequencing of tRNA from Thermophiles

The sequence of tRNA provides the most basic information of tRNA. However, as shown in Figure 2, which displays nucleotide sequences of tRNAs from thermophilic eubacteria [10,11,18,19,20,21,22,23,24,64,67] and archaea [25,36,44,252], the sequences of only 14 tRNA species have been reported from thermophiles. In addition, in the case of Aquifex aeolicus tRNA^Cys, the sequence has been only partially determined [24].

Figure 2.

Sequences of tRNA from thermophiles. The modified nucleosides are indicated in red with their positions. Parentheses indicate that a portion of the modified nucleoside is further modified to its derivative. Abbreviations of modified nucleosides are given in Supplementary Table S1. (A) Geobacillus stearothermophilus tRNA^Leu. (B) G. stearothermophilus tRNA^Phe. (C) G. stearothermophilus tRNA^Tyr. (D) G. stearothermophilus tRNA^Val. (E) Aquifex aeolicus tRNA^Cys. (F) Thermus thermophilus tRNA^Asp. (G) T. thermophilus tRNA^Ile. (H) T. thermophilus tRNA^Met_f1. (I) T. thermophilus tRNA^Phe. (J) Thermoplasma acidophilum initiator tRNA^Met. (K) T. acidophilum elongator tRNA^Met. (L) T. acidophilum tRNA^Leu. (M) Sulfolobus acidocaldarius initiator tRNA^Met In A. aeolicus tRNA^Cys (E) the nucleotides shown in gray could not be determined and cyanoethylated tRNA^Cys was not analyzed. Therefore, this tRNA may possess additional modifications (e.g., Ψ₃₉, Ψ₅₅ and m¹A58). Thermus thermophilus possesses two tRNA^Met_f species. The difference of tRNA^Met_f2 is single G-C base pair, which is indicated in purple in (H). In S. acidocaldarius initiator tRNA^Met (M), the nucleosides at positions 9 and 26 may be m¹A₉ and m²₂Gm₂₆, respectively.

In general, sequencing of tRNA from thermophiles is difficult for the following reasons. First, purification of specific tRNA from thermophiles is not easy. Currently, tRNA is purified by the solid DNA probe method [298,299,300]. In this method, the solid-phase complementary DNA probe is placed in a column and hybridized with the target tRNA and then the target tRNA is eluted from the column. Since the structures of tRNA from thermophiles are very rigid, denaturing the tRNA to allow hybridization is difficult. This problem has been solved by incorporating tetraalkyl-ammonium salt in the hybridization buffer [301]. This salt destabilizes the secondary and tertiary structures of tRNA and promotes formation of the RNA-DNA hetero-duplex. This alteration enabled us to purify A. aeolicus tRNA^Cys [24], Thermus thermophilus tRNA^Phe [11], tRNA^Met_f1 [248] and tRNA^Thr [263,302], Thermoplasma acidophilum initiator tRNA^Met [89], elongator tRNA^Met [89], and tRNA^Leu [36]. Even with the use of tetraalkyl-ammonium salt, however, the solid DNA probe method is not versatile. For example, because the difference between T. thermophilus tRNA^Met_f1 and tRNA^Met_f2 is only one G-C base pair in the T-stem (Figure 2H) [21], purification of tRNA^Met_f1 required its separation from tRNA^Met_f2 by BD-cellulose column chromatography before the solid DNA probe method could be applied [248].

Second, since the structure of tRNA from thermophiles is rigid, limited cleavage by formamide [303,304] is difficult. Therefore, it is difficult to apply the classical technique used for RNA sequencing to tRNA from thermophiles. Liquid-chromatography/mass-spectrometry (LC/MS) has been found to be the most reliable method to overcome this problem [305,306]. In general, LC/MS requires prior cleavage of tRNA by RNases. However, because the G-C content in the stem regions of tRNA from thermophiles is very high (Figure 2), RNA fragments with the same sequences are often generated by RNase cleavage. Therefore, use of multiple RNases and/or preparation of gene disruptant strains are required to overcome this problem.

Furthermore, given that it is not possible to distinguish uridine and pseudouridine by MS, cyanoethylation of tRNA is generally required to detect this nucleoside [307]. In the sequencing of T. acidophilum tRNA^Leu [36], we used a combination of the cyanoethylation and classical formamide method for detection of Ψ₅₄ because the efficiency of cyanoethylation of Ψ₅₄ was low. Thus, specific techniques are required even if an LC/MS system is available.

Third, to determine the modified nucleoside precisely, preparation of a standard compound is often required. For example, it was necessary to prepare the standard ncm⁵U nucleoside from the Saccharomyces cerevisiae trm9 gene disruptant strain [308] to determine the anticodon modification of T. acidophilum tRNA^Leu [36]. In some cases, synthesis of a standard compound by organic chemistry may be required. Lastly, preparing cultures of thermophiles is not so easy for general biochemical researchers (e.g., under anaerobic conditions at high temperatures).

To overcome these problems, the cooperation of researchers in different fields is required. At present, the solid DNA probe method with tetraalkyl-ammonium coupled with LC/MS is the main method for sequencing tRNA from thermophiles. Therefore, it is anticipated that a large numbers of sequences of tRNA from thermophiles will be reported by using this approach in the future.

3. Modified Nucleosides in tRNA from Moderate Thermophiles Are Common to Those from Mesophiles

Seven tRNA sequences from moderate thermophiles (Geobacillus stearothermophilus and T. acidophilum), which live at below 75 °C, have been reported (Figure 2). Furthermore, the modified nucleosides in unfractionated tRNA from moderate thermophiles (Methanobacterium thermoaggregans, Methanobacterium thermoautotrophicum, and Methanococcus thermolithotrophicus) were analyzed [97,99]. These studies have shown that the modified nucleosides in tRNA from moderate thermophiles are typically common to those in tRNA from mesophiles. In summarizing the information on tRNA modifications and tRNA modification enzymes by thermophilic species [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330] (Table 2), we have separately considered moderate thermophiles, extreme-thermophiles, and hyper-thermophiles. However, there are some differences between moderate thermophiles and mesophiles. For example, the degree of 2’-O-methylation in tRNA from G. stearothermophilus is increased at high temperatures [309]. Furthermore, several modifications (Gm₁₈, D modifications, and Gm₃₄) in tRNA from G. stearothermophilus cannot be explained by the enzymatic activities of the already-known tRNA modification enzymes, which is described in Table 2. Moreover, T. acidophilum possesses several distinct tRNA modifications such as G⁺₁₃ and m⁷G₄₉ (Table 2) [36]. Although these differences are present, thermophile-specific modified nucleosides have not been found in tRNA from moderate thermophiles, which suggests that living organisms can survive at 75 °C via the tRNA modifications in mesophiles.

Table 2.

Thermophiles: their tRNA modifications and tRNA modification enzymes.

Species	Predicted Enzyme	Distinct tRNA Modifications and General Information	References
Moderate Thermophiles
Eubacteria
Geobacillus stearothermophilus (Bacillus stearothermophilus) 30–75 °C		Sequences of tRNALeu [18], tRNAPhe [19], tRNATyr [20], and tRNAVal2 [64] have been reported (Figure 2). The majority of modifications in tRNA are similar to those in B. subtilis. With increasing culture temperature, the extent of 2’-O-methylation in the tRNA mixture increases [309].
	Gm18 (TrmH?)	Although trmH is not encoded in the B. subtilis genome, a trmH-like gene is encoded in the G. stearothermophilus genome. Gm18 has been found in tRNATyr but not in tRNALeu. This modification pattern suggests that the substrate tRNA specificity of G. stearothermophilus TrmH may be different from that of other known TrmH enzymes.	[20]
	D17, D20 and D20a (Dus family protein?)	In G. stearothermophilus tRNA, D17, D20, and D20a modifications have been reported. In E. coli, three Dus family proteins known as DusA, DusB, and DusC, produce D20 and D20a, D17, and D16, respectively [65,66]. In the G. stearothermophilus genome, however, only one gene is annotated as a dus-like gene. Therefore, D modifications in G. stearothermophilus cannot be explained by the tRNA substrate specificity of the known Dus proteins.	[17,19,64]
	m1A22 (TrmK?)	The m1A22 modification has been found in tRNATyr and tRNASer from B. subtilis and Mycoplasma capricolum [310,311]. G. stearothermophilus tRNALeu and tRNATyr possess m1A22 [18,20]. The presence of a trmK-like gene in the genome of G. stearothermophilus has been reported [86].	[20,86]
	Gm34 (unknown MT)	G. stearothermophilus tRNAPhe possesses Gm34 (Figure 2B) [19]. In contrast, the nucleoside at position 34 in E. coli tRNAPhe is unmodified G. Given that E. coli TrmL acts only on tRNALeu isoacceptors [143], the 2′-O-methylation of G34 in tRNAPhe from G. stearothermophilus is cannot be simply explained by the activity of known TrmL.	[19]
	m6A37 (YfiC; TrmG?)		[198]
Archaea
Methanobacterium thermoaggregans Optimum growth temperature 60 °C		Sequences of tRNAAsn and tRNAGly have been reported [8].
Methanobacterium thermoautotrophicum 45–75 °C		The modified nucleosides in unfractionated tRNA are essentially common to those in tRNA from mesophilic methane archaea [97].
Methanococcus thermolithotrophicus 17–62 °C		The modified nucleosides in unfractionated tRNA are essentially common to those in tRNA from mesophilic methane archaea [99].
Thermoplasma acidophilum Optimum growth temperature 55–60 °C		Sequences of tRNAMeti [44,252], tRNAMetm [25], and tRNALeu [36] have been reported. Several recombinant tRNA modification enzymes have been used for biochemical studies.
	s4U8 and s4U9 (ThiI? + α)	The s4U9 modification has been found in tRNALeu [36]. The sulfur donor for s4U formation is unknown [35].	[36]
	G+13 and G+15 (ArcTGT + ArcS?)	The G+13 modification has been found only in tRNALeu from T. acidophilum [36]. ArcTGT from T. acidophilum acts on both G13 and G15 in tRNALeu [47].	[36,47]
	m22G26 (Trm1)		[89]
	ncm5U34 (Elp3?)		[36]
	m1G37 (Trm5)		[89]
	m7G49 (unknown MT)		[36]
	Cm56 (Trm56)	The presence of unusual trm56-like gene in the T. acidophilum genome has been reported in a bioinformatics study [250]. The Trm56 enzymatic activity has been confirmed via the recombinant protein [89]. T. acidophilum Trm56 exceptionally possesses a long C-terminal region in the SPOUT tRNA MT [312].	[89,250,312]
Extreme-thermophiles and Hyper-thermophiles
Eubacteria
Aquifex aeolicus Optimum growth temperature 85–94 °C		The partial sequence of tRNACys has been reported [24] (Figure 2E). Several tRNA MT activities have been detected in the A. aeolicus cell extract using an E. coli tRNA mixture [24]. The tRNA modification enzymes listed below were characterized via recombinant proteins.
	Gm18 (TrmH)		[74,77]
	D20 (Dus)	D20 exists in tRNACys. However, the nucleoside at position 20a is unmodified U [24]. Therefore, A. aeolicus Dus may act only on U20.	[24,82]
	m2G26, m22G26, m2G27 and m22G27 (Trm1)	Aquifex aeolicus exceptionally possesses Trm1 in eubacteria [24].	[24,95]
	I34 (TadA)		[104,105]
	mnm5U34 (MnmC2)	MnmC catalyzes the final methylation step of mnm5U synthesis. Aquifex aeolicus MnmC2 comprises only an MT domain.	[136]
	(MnmD; previously called GidA)		[140,141]
	k2C34 (TilS)		[111,112,113]
	m1G37 (TrmD)	The dimer structure of A. aeolicus TrmD is stabilized by inter-subunit disulfide bonds [165].	[160,162,165]
	m7G46 (TrmB)	TrmB proteins from thermophiles (A. aeolicus and T. thermophilus) possess a long C-terminal region.	[206,207,208]
	m5U54 and m5s2U54 (TrmFO)	The presence of trmFO gene in A. aeolicus genome was initially described in Reference [221].	[24,221]
	m1A58 (TrmI)		[257,262]
Thermotoga maritima 80–90 °C		Sequences of tRNA from T. maritima have not been reported. Recombinant proteins have been used for biochemical and structural studies.
	hn6A (?)	hn6A was first identified in modified nucleosides from unfractionated tRNA from T. maritima [313]. Because hn6A was subsequently found in modified nucleosides from psychrophilic archaea [56], it is not a thermophile-specific modification. Thermotoga maritima and Thermodesulfobacterium commune exceptionally possess hn6A in eubacteria. The modification position in tRNA, modified tRNA species, and biosynthesis pathway of hn6A are unknown.	[56,313]
	s4U8 (ThiI + IscS)		[31,32]
	oQ34 (QueA)		[151]
	mnm5U34 (TrmE)		[138,139]
	t6A37 (TsaB, TsaC/TsaC2, TsaD and TsaE)		[190]
	ms2i6A37 (MiaB)		[194,195,196]
	m1G37 (TrmD)		[171]
	m5U54 and m5s2U54 (TrmFO and TtuA)	The m5s2U nucleoside has been found in unfractionated tRNA from T. maritima [97].	[97,134,221,222]
	Ψ55 (TruD)		[244,245,246,247]
	m1A58 (TrmI)		[257]
Thermodesulfobacterium commune Optimum growth temperature 70 °C	hn6A and ms2hn6A (?)	hn6A and ms2hn6A have been found in modified nucleosides from unfractionated tRNA from T. commune. The ms2hn6A modification may be derived from hn6A. So far, T. commune is the only eubacterium that possesses ms2hn6A in tRNA. The modification position in tRNA, modified tRNA species, and biogenesis pathway of hn6A and ms2hn6A are unknown.	[313]
Thermus flavus Optimum growth temperature 70 °C		Partial purification of tRNA m1A58 MT has been reported: the activity of tRNA m7G46 MT has also been described [204].
Thermus thermophiles 50–83 °C		Sequences of tRNAMetf1 [21], tRNAMetf2 [21], tRNAIle1 [67], tRNAAsp [23], and tRNAPhe [10,11] have been reported (Figure 2). Partial sequences of tRNASerGGA [259], tRNAProGGG [314], and tRNAProGGA [314] have been determined. The modification extent of Gm18, m5s2U54 and m1A58 changes with the culture temperature. At high temperatures (>75 °C), m7G46 [11], m5s2U54 [230], and m1A58 [260] modifications are essential for survival. At low temperatures (<55 °C), Ψ55 is essential for survival [248] and m5U54 supports this effect [225] (see the main text). Recombinant proteins have been used for biochemical and structural studies.
	m2G6 (TrmN)		[10,11,13,14,15]
	Gm18 (TrmH)		[10,11,21,23,30,69,70,71,72,73,75,76,78,79,80,81]
	D20 and D20a (DusA)		[10,11,23,67,83,84,85]
	Cm34 and cmnm5Um34 (TrmL)		[144]
	Ψ39 and Ψ40 (TruA)		[10,11,18,23,202]
	m7G46 (TrmB)		[10,11,21,23]
	m5U54 and m5s2U54 (TrmFO + TtuA + TtuB + TtuC + TtuD + IscS)		[10,11,17,21,23,67,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,239]
	Ψ55 (TruB)		[10,11,21,23,67,248]
	m1A58 (TrmI)		[11,30,257,259,260,261,263,264]
Archaea
Aerophyrum pernix 80–100 °C	Ψ13 and Ψ15 (archaeal Pus7 and H/ACA guide RNA system)	A guide RNA for Ψ formation has been predicted based on genome sequencing.	[46]
Archaeoglobus fulgidus 60–95 °C		Modified nucleosides in unfractionated tRNA from A. fulgidus have been reported [97].
	agm2C34 (TiaS)		[116,118,119,315]
Methanocaldcoccus igneus (Methanococcus igneus; Methanotorris igneus) 45–91 °C		Modified nucleosides in unfractionated tRNA from M. igneus have been reported [56,99].
Methanocaldococcus infernus 55–92 °C	cm5U34 (Elp3)		[142]
Methanocaldcoccus jannashii (Methanococcus janaschii) 48–94 °C		Although sequences of tRNA are unknown, the recombinant proteins listed below have been used for biochemical and structural studies.
	m2G6 (Trm14)		[12]
	G+15 (ArcTGT + ArcS)		[51,59]
	Cm34 and Um39 (L7Ae, Nop5, aFib, Box C/D guide RNA system)		[316]
	m1G37 (Trm5)		[159,161,163,164,166,167,168,169,170]
	imG237 (Trm5b + Taw1)		[173,179]
	yW-8637 (Taw2)		[174]
	m5C48 and m5C49 (archaeal Trm4)		[210]
	Ψ54 and Ψ55 (Pus10)		[211,212,213,214]
	m1Ψ54 (Pus10 + TrmY)		[215,216,217]
	Ψ55 (archaeal Cbf5)		[240]
Methanopyrus kandleri 84–110 °C (Strain 116: up to 122 °C)		Many unique modified nucleosides have been found in unfractionated tRNA [100]. tRNAs likely contain many 2’-O-methylated nucleosides derived from the C/D box guide RNA system [17].
	ac6A (?)	The ac6A nucleoside has been purified from the modified nucleosides in unfractionated tRNA and its structure determined. The modification site, modified tRNA species, and biosynthesis pathway are unknown.	[100]
	U8 (CDAT8)		[16]
Methanothermus fervidus 80–97 °C		Only tRNA genes were reported in an early study [317].
Nanoarchaeum equitans 70–98 °C		A unique tRNA processing system has been found [318,319]. The processing of small RNAs in N. equitans is reviewed in Reference [320].
	m1G37 and imG237 (Trm5a)		[176]
	m5U54 (TrmA-like protein)		[237]
Pyrobaculum aerophilum Optimum growth temperature 100 °C	Cm56 (L7Ae, Nop5, aFib, Box C/D guide RNA system)	Cm56 in tRNA is generally produced by Trm56. However, this modification in P. aerophilum is synthesized by the C/D box guide RNA system.	[249]
Pyrobaculum calidifontis 90–95 °C	G+15 (ArcTGT + QueF-like protein)	Eubacterial QueF catalyzes the conversion from preQ0 to preQ1. In P. caldifontis, however, QueF-like protein catalyzes the conversion from preQ0 at position 15 in tRNA to G+15.	[60,62,63]
Pyrobaculum islandicum Optimum growth temperature 100 °C		Modified nucleosides in unfractionated tRNA from P. islandicum have been reported [97].
Pyrococcus abyssi Optimum growth temperature 96 °C		No tRNA sequence has been determined. However, the tRNA modification enzymes listed below have been characterized.
	m2G10 and m22G10 (archaeal Trm11, Trm-G10 enzyme, Trm-m22G10 enzyme)		[40,41]
	Ψ13 and Ψ35 (archaeal Pus7 and H/ACA guide RNA system)		[46]
	Cm34 and Um39 (L7Ae, Nop5, aFib, and C/D box guide RNA system)	Cm34 and Um39 in tRNATrp are formed by the C/D box guide RNA system in which the intron functions as a guide RNA.	[154,155]
	m1G37 (Trm5b)		[180]
	m1G37 and imG237 (Trm5a)		[176,177,179]
	imG-1437 (Taw1)		[173,175]
	t6A37 (Kae1)		[185]
	(KEOPS complex)		[184]
	(Sua5 + KEOPS complex)		[187,189]
	m5C48 and m5C49 (archaeal Trm4 + archaese)		[209]
	m5U54 (TrmA-like protein, PAB0719)		[237,238]
	Ψ55 (Cbf5 + Nop10)		[241]
	Cm56 (Trm56)		[249]
	m1A57 and m1A58 (archaeal TrmI)		[255,256,257,258]
Pyrococcus furiosus Optimum growth temperature 100 °C		Modified nucleosides in unfractionated tRNA from P. furiosus have been reported [98]. Activity of several tRNA modification enzymes has been detected in the cell extract of P. furious [9].
	m2G6 (Trm14)		[13,15]
	m2G10 and m22G10 (archaeal Trm11, Trm-G10 enzyme, Trm-m22G10 enzyme)		[42]
	G+15 (ArcTGT)		[57]
	m2G26 and m22G26 (Trm1)		[91,92]
	t6A37 (KEOPS complex)		[188]
	Ψ54 and Ψ55 (Pus10)		[212,214]
	Ψ55 (Cbf5 + Nop10 + Gar1)		[242]
Pyrococcus horikoshii 80–102 °C		The crystal structure of Nop5 in the C/D box guide RNA system from P. horikoshii has been solved [321].
	G+15 (ArcTGT)		[50,52,53,54,55,89]
	m2G26 and m22G26 (Trm1)		[89,93]
	yW-8637 (Taw2)		[174]
	m5s2U54 (TtuA)		[233]
	Cm56 (Trm56)		[251]
Pyrodictium occultum Optimum growth temperature 105 °C		Modified nucleosides in unfractionated tRNA have been analyzed and many 2’-O-methylated nucleosides found [97,98]. mimG was originally found among the modified nucleosides in tRNAs from P. occultum, Sulfolobus solfaraticus, and Thermoproteus neutrophilus [322]. Although the melting temperature of P. occultum tRNAMeti transcript is only 80 °C and that of native tRNAMeti is more than 100 °C (see main text) [323].
Pyrolobus fumarii This archaeon can survive at 113 °C.		Modified nucleosides in unfractionated tRNA have been analyzed [324].
Stetteria hydrogenophila Optimum growth temperature 95 °C		Modified nucleosides in unfractionated tRNA have been analyzed and methyl-hn6A, ms2hn6A, and m2, 7Gm identified [56].
Sulfolobus acidocaldarius Optimum growth temperature 75–80 °C		Sequence of tRNAMeti has been reported [44]. The m1I57 modification was originally found in tRNAs from S. acidocaldarius and Haloferax volcanii [253]. G+ was first isolated from the nucleosides in S. acidocaldarius tRNAs and its structure determined [49]. The structures of wyosine derivatives (imG-14 and imG2) have been determined by using the nucleosides from S. acidocaldarius tRNAs [325].
	m1A9 (archaeal Trm10)		[37,38]
	Ψ13 and Ψ35 (archaeal Pus7 and H/ACA guide RNA system)		[46]
	Cm32 (archaeal TrmJ)		[96]
Sulfolobus solfaraticus 55–90 °C		mimG was originally found among the modified nucleosides in tRNAs from P. occultum, S. solfaraticus, and Thermoproteus neutrophilus [322]. The structure of box C/D RNP from S. solfaraticus has been reported [326].
	agm2C (TiaS)	The identification of agm2C34 in Haloarcula marismortui tRNAIle2 and the presence of agm2C in S. solfaraticus tRNA have been reported.	[117]
	Ψ13 and Ψ35 (archaeal Pus7 and H/ACA guide RNA system)	Generally, Ψ35 in tRNATyr is synthesized by archaeal Pus7. However, Pus7 from S. solfaraticus possesses weak Ψ13 formation activity but not Ψ35 formation activity. In S. solfaraticus and A. pernix, a guide RNA for Ψ35 formation exists.	[46]
	imG237 (Trm5a; SSO2439 protein)	Trm5a (SSO2439 protein) does not possess m1G37 formation activity and is used only for imG2 formation.	[178]
	mimG37 (Taw3)		[180]
Sulfolobus tokodaii This archaeon can survive at 87 °C.	Ψ13 and Ψ35 (archaeal Pus7 and H/ACA guide RNA system)		[46]
	t6A37 (Sua5)		[327,328,329]
Thermococuus celer This archaeon can survive at 85 °C.		Although tRNA genes were analyzed in an early study [330], there is no information on tRNA modifications.
Thermococcus kodakarensis (Thermococcus kodakaraensis; Pyrococcus kodakarensis) 65–100 °C	m1A9 and m1G9 (archaeal Trm10)		[37,39]
	m2G10 and m22G10 (archaeal Trm11, Trm-G10 enzyme, Trm-m22G10 enzyme)		[43]
	G+15 (ArcTGT)		[47]
	m5U54 (TrmA-like protein)		[237]
Thermoproteus neutrophilus Optimum growth temperature 85 °C		Modified nucleosides in unfractionated tRNA have been analyzed [97]. mimG was originally found among the modified nucleosides in tRNAs from P. occultum, S. solfaraticus, and T. neutrophilus [322].

Only distinct modifications that have been investigated are listed by thermophile species. In many cases, only tRNA modification enzymes (rather than modifications) have been studied by using recombinant proteins. For example, the presence of the m⁷G₄₆ modification has not been confirmed in tRNA from A. aeolicus, but TrmB (tRNA m⁷G₄₆ MT) has been characterized through the recombinant protein. In this case, m⁷G₄₆ (TrmB) is listed in the section “Aquifex aeolicus”. The moderate thermophiles and extreme-thermophiles along with hyper-thermophiles are separated. Transfer RNA modifications in thermophilic eukaryotes are unknown. Abbreviation: MT, methyltransferase.

4. Strategies of tRNA Stabilization by Modified Nucleosides in Extreme-Thermophiles and Hyper-Thermophiles

In general, the G-C content in the stem regions of tRNA from thermophiles is very high (Figure 2). However, the stability of tRNA from thermophiles cannot be explained only by the increase in G-C content in the stem region. For example, although the melting temperature of T. thermophilus tRNA^Phe transcript is 76 °C, that of the native tRNA^Phe is 84.5 °C [11]. Thus, modified nucleosides are essentially required for stabilization of tRNA at high temperatures. Modified nucleosides in tRNA from thermophiles have been studied mainly from the view point of tRNA stabilization. So far, only a few modified nucleosides specific to thermophiles have been found (Figure 3). These thermophile-specific modified nucleosides seem to stabilize the tRNA structures at high temperatures. As described below, extreme-thermophiles and hyper-thermophiles possess two strategies of tRNA stabilization by modified nucleosides. One is based on thermophile-specific modification such as m⁵s²U₅₄ (Figure 3A) and the other is based on 2′-O-methylations at multiple positions in tRNA (Figure 3B–E). Recently, the unknown modified nucleoside at position 26 in Sulfolobus acidocaldarius tRNA^Met (Figure 2M) was described as m²₂Gm [96]. On the whole, however, the modification site(s), modified tRNA species, and biosynthesis pathways of most thermophile-specific modified nucleosides are unknown. Moreover, these nucleosides may have additional functions at high temperatures beyond their structural effect.

Figure 3.

Thermophile-specific modified nucleosides in tRNA. Abbreviations of modified nucleosides are given in Supplementary Table S1. (A) m⁵s²U. (B) m⁵Cm. (C) m¹Im. (D) m²₂Gm. (E) m^{2, 7}Gm. The modifications are indicated in red.

4.1. m⁵s²U₅₄ Is a Typical Thermophile-Specific Modified Nucleoside in tRNA

The m⁵s²U₅₄ modification was originally found in tRNA from T. thermophilus [331]. Subsequently, this modified nucleoside was found in tRNA from A. aeolicus, T. maritima, Pyrococcus abyssi, Pyorococcus horikoshii, and T. kodakarensis (Table 2) but not from mesophiles. The m⁵s²U₅₄ modification forms a reverse Hoogsteen base-pair with A₅₈ (or m¹A₅₈) in tRNA and stabilizes the tRNA structure by stacking with the G₅₁–C₆₁ base-pair [220]. Because the 2-thio-modification at position 54 increases the melting temperature of tRNA by more than 3 °C [22,218,220], the m⁵s²U₅₄ modification contributes to stabilization of the tRNA structure. The degree of m⁵s²U₅₄ modification increases with an increasing temperature [22,67,220,229]. At 80 °C, the extent of m⁵s²U₅₄ modification in tRNA is almost 100% [22,67,220,229]. The melting temperature of tRNA mixture is maintained above 85 °C due to the presence of m⁵s²U₅₄ modification [229] and T. thermophilus can grow at 50 to 83 °C. Thus, living organisms can survive at 80 °C due to the presence of m⁵s²U₅₄ modification in tRNA.

4.2. The Network Between Modified Nucleosides in tRNA and tRNA Modification Enzymes in T. thermophilus Adapts Protein Synthesis at Low and High Temperatures

Under natural conditions, the temperature of hot spring water fluctuates for several reasons including an influx of river water, snowfall, and an eruption of hot water. In accordance with these temperature changes, T. thermophilus can synthesize proteins efficiently at a wide range of temperatures (50 to 83 °C) by regulating the flexibility (rigidity) of its tRNA [220]. At high temperatures (above 75 °C), three modified nucleosides in tRNA, m⁵s²U₅₄ [230], m¹A₅₈ [260], and m⁷G₄₆ [11] are essential for survival of T. thermophilus. The m¹A₅₈ modification is one of the positive determinants for the two-thiolation system of m⁵s²U₅₄. Thus, a T. thermophilus disruptant strain of the trmI gene encoding the tRNA m¹A₅₈ methyltransferase cannot grow at 80 °C [229,260]. The presence of m⁷G₄₆ modification in tRNA increases the speed of tRNA modification enzymes such as TrmH for Gm₁₈, TrmD for m¹G₃₇, and TrmI for m¹A₅₈ [11]. The m¹A₅₈ modification further increases the rate of sulfur-transfer to m⁵U₅₄ by the 2-thiolation system and the introduced modified nucleosides coordinately stabilize the tRNA structure. Thus, the m⁷G₄₆ modification produced by TrmB is a key factor in the network between modified nucleosides in tRNA and tRNA modification enzymes of T. thermophilus at high temperatures. In the trmB-gene disruptant starin, tRNA^Phe and tRNA^Ile were found to be degraded by a temperature shift from 70 °C to 80 °C and heat-shock proteins were not synthesized efficiently [11].

At low temperatures (below 55 °C), in contrast, the Ψ₅₅ modification produced by TruB is essential for the survival of T. thermophilus [248]. The presence of Ψ₅₅ stabilizes both the local structure of the T-arm and the interaction of the T-arm with the D-arm in tRNA. The local rigidity in tRNA caused by Ψ₅₅ slows down the speeds of introducing modified nucleosides around Ψ₅₅ (Gm₁₈, m⁵s²U₅₄ and m¹A₅₈), which maintains the flexibility of tRNA at low temperatures. The presence of m⁵U₅₄ modification by TrmFO supports this effect of Ψ₅₅ [225].

It should be mentioned that D modifications are thought to bring flexibility to tRNA because D does not stack with other bases and brings about the C2′-endo form of ribose [332]. However, a T. thermophilus disruptant strain of the dusA gene encoding tRNA D₂₀/D_20a synthase did not show growth retardation at 50, 60, 70, or 80 °C, and abnormal modifications were not observed in tRNA from this strain [85]. Therefore, the function of D₂₀ and D_20a modifications is unknown. Since DusA recognizes the interaction of T-arms and D-arms in tRNA [84], the stabilization of the L-shaped tRNA structure by other modified nucleosides is required for the efficient introduction of D₂₀ and D_20a at high temperatures [85]. Thus, D₂₀ and D_20a are relatively late modifications in T. thermophilus tRNA.

Although the above network is a temperature adaptation system of T. thermophilus, it regulates the order in which modified nucleosides are introduced into tRNA. Similar networks have been found in mesophiles [333]. In Escherichia coli, for example, the 2′-O-methylation at position 34 by TrmL requires an i⁶A₃₇ modification [334]. However, the network in T. thermophilus is distinct because it regulates the structure of a three-dimensional core and many modifications in tRNA are related. One of the advantages of this system is that protein synthesis is not required. The response of the system is very rapid. It is possible that thermophilic archaea possess a similar network between modified nucleosides in tRNA and tRNA modification enzymes because some of them can also grow at a wide range of temperatures.

4.3. Stabilization of tRNA Structure by 2′-O-Methylation

Because 2′-O-methylation shifts the equilibrium of ribose puckering to the C3′-endo form and enhances the hydrophobic interaction, this modification, when carried out at multiple positions, brings rigidity of tRNA. Furthermore, 2’-O-methylations prevents hydrolysis of phophodiester-bonds in tRNA at high temperatures. Therefore, 2’-O-methylations may prolong the half-lives of tRNA. Notably, there is a living organism in which tRNA is stabilized without m⁵s²U₅₄ modification. A hyper-thermophilic archaeon, Pyrodictium occultum can grow at 105 °C, and various 2′-O-methylted nucleosides such as Ψm, m¹Im, and m²₂Gm are present in its tRNA, but s²U and m⁵s²U are not observed [97,98]. Notably, although the melting temperature of the P. occultum tRNA^Met transcript is 80 °C, that of the native tRNA^Met is more than 100 °C [323]. Thus, the melting temperature of P. occultum tRNA is increased by more than 20 °C through a combination of numerous 2′-O-methylated nucleosides.

Methanopyrus kandleri can grow at more than 110 °C and tRNAs from this archaeon contain many unique modifications such as U₈ (the product of C₈ to U₈ editing) [16], ac⁶A, m^{2, 7}Gm, and methyl-hn⁶A [100]. Furthermore, M. kandleri possesses 132 species of C/D-box guide RNAs [17], which suggests that RNAs are highly methylated by the L7Ae, Nop5, aFib, and C/D-box guide RNA system. In the case of M. kandleri, therefore, tRNA seems to be stabilized by unique modifications and 2′-O-methylations.

These observations suggest that living organisms can survive at more than 100 °C by a combination of 2′-O-methylations and other thermophile-specific tRNA modifications.

4.4. Other tRNA Stabilization Factors

RNA binding proteins, polyamines, magnesium ions, and potassium ions are all able to stabilize tRNA in thermophiles. For example, transfer RNA-binding protein 111 (Trbp111) is an RNA-binding protein that is observed only in A. aeolicus [335,336,337]. A. aeolicus can grow at 94 °C and modified nucleosides in tRNA of this hyperthermophilic eubacterium are not so different from those in tRNA from T. thermophilus, which grows at temperatures below 83 °C. Therefore, Trbp111 may provide more than 10 °C of tRNA stabilization in A. aeolicus. The docking model of Trbp111 and tRNA suggests that Trbp111 stabilizes the three-dimensional core of tRNA [336]. Archease is another tRNA-binding protein that can change the methylation site of P. abyssi Trm4 [209]. Furthermore, archease promotes the ligation of tRNA exons during tRNA splicing [338,339]. Therefore, it has the potential to stabilize the tRNA structure at high temperatures.

Many tRNA-binding proteins and RNA chaperone proteins have been identified in eukaryotic cells [340,341]. Although these types of protein are unknown in thermophilic eukaryotes, some of them may stabilize the tRNA structure (or help correct folding of tRNA) at high temperatures. Recently, it was revealed that E. coli TruB (tRNA Ψ₅₅ synthase [243]) possesses an RNA chaperone activity [342,343]. In the case of T. thermophilus, although the Ψ₅₅ modification is required for survival at low temperatures (below 55 °C), the truB gene disruptant strain shows abnormal growth at 80 °C [248]. Therefore, the RNA chaperone effect of TruB may also be expressed at high temperatures in T. thermophilus. Furthermore, these observations suggest that other tRNA modification enzymes have the potential to work as RNA chaperones.

In general, polyamines have the potential to interact with nucleic acids and phospholipids because they possess multiple positive charges and hydrophobic areas. There are several studies on the interaction between tRNA and polyamines [344,345,346,347]. Thermophiles produce unique polyamines including long and branched polyamines [348,349,350,351]. Therefore, polyamines probably contribute to stabilize the tRNA structure at high temperatures. Furthermore, in vitro studies have shown that thermophile-specific long and branched polyamines affect the activities of several tRNA modification enzymes [81,352]. For example, TrmH from T. thermophilus methylates tRNA transcript at 80 °C only in the presence of long or branched polyamines [81]. Moreover, the long and branched polyamines are required for the maintenance of several tRNAs and the 70S ribosome and are essential for the survival of T. thermophilus at high temperatures [353].

Lastly, magnesium ions have been shown to be a tRNA stabilization factor [6,88,354] and are very important when considering the structural effects of several modified nucleosides in tRNA [58,88,354,355,356]. However, the precise concentration of magnesium ions in thermophile cells is unknown. It may differ depending on the growth environments. Potassium ions also function as RNA stabilization factor [88]. Notably, the interacellular concentration of some hyperthermophilic archaea (M. fervidus and P. furiosus) is much higher (700–900 mM) than that of mesophilic archaea [357]. In the case of Methanothermus sociabilis, the interacellular potassium concentration reaches 1060 mM [357]. These high concentrations of potassium ions may have effects on the stability of tRNA and the activities of tRNA modification enzymes.

5. tRNA Modifications and Environmental Stresses at High Temperatures

Recent studies have revealed that the modifications in tRNA are stress-resistance and/or stress-response factors [102,358,359,360,361]. Furthermore, a high temperature itself can be a stress factor for living organisms because some modified nucleosides (D and m⁷G) are liable at high temepratures [297].

5.1. Oxidative Stress

Many thermophiles can grow under aerobic conditions. For example, Aerophyrum pernix can grow at 100 °C under aerobic conditions. Under such conditions at high temperatures, living organisms seem to be exposed to heavy oxidative stress, which is a typical environmental stress. The amount of antioxidant enzymes such as superoxide-dismutase, catalase, and peroxidase in Thermus filiformis, which is an extreme-thermophilic eubacterium, increases at high temperatures [362].

Among tRNA modification enzymes, both Fe-S cluster proteins [34,130,134,142,150,173,196,236,363] for sulfur-transfer, reduction of base and/or radical S-adenosyl-l-methionine (SAM) reaction, and enzymes with catalytic cysteine residues [141,210,364,365,366], seem to be easily changed under oxidative stress. In some cases, the substrate (e.g., electron donors and folate derivatives [126,221,227,367]) may be unstable under aerobic conditions at high temperatures. Similarly, several modified nucleosides such as D and s⁴U may be labile under oxidative stress at high temperatures. Therefore, aerobic thermophiles need to protect their cellular components from oxidative stress and their tRNA modifications may respond to such stress as in mesophiles. Overall, however, the relationship between oxidative stress and tRNA modifications in thermopiles is unclear. In addition, tRNA modification systems in some thermophiles may utilize aerobic conditions at high temperatures. For example, A. aeolicus grows under microaerophilic conditions at high temperatures (80–94 °C) and the dimer structure of A. aeolicus TrmD is stabilized by inter-subunit disulfide bonds [165].

5.2. Other Environmental Stresses

Thermophiles often live in severe environments such as extreme pH and high pressure in addition to high temperatures. These environmental stresses may give rise to the diversity of tRNA modifications. At present, however, there are no data to support this viewpoint.

UV-stress is one such environmental stress and the s⁴U modification in tRNA is a known UV-stress-resistance factor for E. coli [368] and Salmonella typhimurium [27]. Thus, the s⁴U modification in tRNA is likely to work similarly to a UV-resistant factor in thermophiles. Interestingly, the genomes of Archaeoglobus fulgidus and Methanocaldococcus janaschii, which were isolated from the oil mines under the sea and deep sea, respectively, contain a thiI genes [369] (AF_RS04455 and MJ_RS04985, respectively) encoding tRNA s⁴U₈ synthetase. Since sunlight does not reach the environments in which these thermophilic archaea live, the s⁴U modification and/or ThiI may have an additional function (e.g., sulfur-metabolism) in these archaea. Furthermore, it was recently reported that the melting temperature of tRNA from an E. coli thiI-gene disruptant strain was decreased relative to the wild-type strain [33]. Therefore, the s⁴U₈ modification may contribute to stabilize tRNA structure. Furthermore, UV-stress may have an effect on other tRNA modifications via the cross-linking of s⁴U in tRNA. For example, the methylation speed of T. thermophilus TrmH is decreased when the substrate tRNA is cross-linked [30].

Lastly, the availability of nutrient-factors may have an effect on tRNA modifications in thermophiles. To test this idea, the extent of modifications in tRNA from T. thermophilus cells cultured in a nutrient-poor condition was investigated [227]. Contrary to expectation, the extent of the modification of all methylated nucleosides analyzed was normal, which demonstrates that the limited nutrients were preferentially consumed in the tRNA modification systems [227]. Thus, the findings indicated the importance of tRNA modifications for the survival of T. thermophilus.

6. Utilization of tRNA Modification Enzymes from Thermophiles

Given that proteins from thermophiles are heat-resistant and very stable, numerous tRNA modification enzymes have been used in biochemical and structural studies (Table 1 and Table 2). In particular, crystal structural studies of thermostable enzymes provided significant information on catalytic mechanisms and RNA-protein interactions. Studies on the crystal structures of tRNA modification enzymes from thermophiles are summarized in Supplementary Table S2. It is anticipated that thermostable proteins will continue to contribute structural studies in the future. Thermostable tRNA modification enzymes can be a tool for molecular and cell biology. For example, A. fulgidus TiaS with agmatine analogues has been used for site-specific RNA-labeling in mammalian cells [315]. In addition, thermostable tRNA modification enzymes may be used for healthcare. For example, Gm₁₈ modification in tRNA does not stimulate the Toll-like receptor 7 [287,288] and tRNA with Gm₁₈ alleviates inflammation [288]. Since TrmH from T. thermophilus can methylate all tRNA species [72] and is very stable, it may be useful for preparing tRNAs with Gm₁₈ modifications for tRNA therapy.

7. Perspective

Given that the temperature of ancient Earth was very high relative to that of present-day Earth, thermophiles may be remnants of ancient living organisms. Therefore, studies on tRNA modification enzymes and modified nucleosides in tRNA from thermophiles will contribute to the considerations of the evolutionary pathways of living organisms. Furthermore, such studies will continue to shed light on the variety and environmental adaptations of living organisms. Moreover, as outlined above, the thermostable enzymes may be useful as biotechnological and medical tools and may contribute toward the production of valuable materials.

Supplementary Materials

The following are available online at http://www.mdpi.com/2076-2607/6/4/110/s1, Table S1: Abbreviations of modified nucleosides, Table S2: Crystal structural studies on tRNA modification enzymes from thermophiles.

Author Contributions

All authors determined the concept of this review and collected the information from the references. H.H. wrote the manuscript and all authors revised and approved the manuscript.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (16H04763 to H.H.) from the Japan Society for the Promotion of Science (JSPS).

Conflicts of Interest

The authors declare no conflict of interest.