Chie Tomikawa

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N7-methylguanine at position 46 (m7G46) in tRNA is produced by tRNA (m7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m7G46 modification in tRNAPhe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m1G37, suggesting that the m7G46 positively affects their formations. Although the lack of the m7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNAPhe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNAPhe and tRNAIle. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m7G46 modification supports introduction of other modifications.

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

The tRNA recognition mechanism of folate/FAD-dependent tRNA methyltransferase (TrmFO)

Background: RNA modification enzymes select specific RNAs as substrates. Results: A novel assay for folate-dependent tRNA methyltransferase (TrmFO) was developed that clarified positive and negative determinants of TrmFO. Conclusion: TrmFO recognizes a T-arm structure including the U54U55C56 sequence and G53-C61 base pair; A38 prevents incorrect methylation of U32. Significance: Studying how proteins recognize RNA is crucial for understanding RNA maturation processes. The conserved U54 in tRNA is often modified to 5-methyluridine (m5U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m5U54 is produced by folate/FAD-dependent tRNA (m5U54) methyltransferase (TrmFO). TrmFO utilizes N5,N10-methylenetetrahydrofolate (CH2THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [14C]CH2THF was supplied from [14C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m1A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m5U54, m1A58, and s2U54 modifications on m5s2U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme.

Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)

Background: tRNA methyltransferases specifically recognize substrate tRNAs. Results: To clarify the tRNA recognition mechanism of TrmI, three tRNA species and 45 variants were analyzed in vitro and in vivo. Conclusion: TrmI recognizes the aminoacyl stem, variable region, C56, purine 57, A58, and U60 in the T-loop of tRNA. Significance: Our in vitro experimental results explain the regulation of in vivo methylation levels in tRNAs. TrmI generates N1-methyladenosine at position 58 (m1A58) in tRNA. The Thermus thermophilus tRNAPhe transcript was methylated efficiently by T. thermophilus TrmI, whereas the yeast tRNAPhe transcript was poorly methylated. Fourteen chimeric tRNA transcripts derived from these two tRNAs revealed that TrmI recognized the combination of aminoacyl stem, variable region, and T-loop. This was confirmed by 10 deletion tRNA variants: TrmI methylated transcripts containing the aminoacyl stem, variable region, and T-arm. The requirement for the T-stem itself was confirmed by disrupting the T-stem. Disrupting the interaction between T- and D-arms accelerated the methylation, suggesting that this disruption is included in part of the reaction. Experiments with 17 point mutant transcripts elucidated the positive sequence determinants C56, purine 57, A58, and U60. Replacing A58 with inosine and 2-aminopurine completely abrogated methylation, demonstrating that the 6-amino group in A58 is recognized by TrmI. T. thermophilus tRNAGGUThrGGUThr contains C60 instead of U60. The tRNAGGUThr transcript was poorly methylated by TrmI, and replacing C60 with U increased the methylation, consistent with the point mutation experiments. A gel shift assay revealed that tRNAGGUThr had a low affinity for TrmI than tRNAPhe. Furthermore, analysis of tRNAGGUThr purified from the trmI gene disruptant strain revealed that the other modifications in tRNA accelerated the formation of m1A58 by TrmI. Moreover, nucleoside analysis of tRNAGGUThr from the wild-type strain indicated that less than 50% of tRNAGGUThr contained m1A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns.

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo‐acidophilic archaeon. We purified tRNALeu (UAG) from T. acidophilum using a solid‐phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m7G‐specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m7G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m7G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4‐thiouridine9, archaeosine13 and 5‐carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.

The C-terminal region of thermophilic tRNA (m7G46) methyltransferase (TrmB) stabilizes the dimer structure and enhances fidelity of methylation.

Transfer RNA (m7G46) methyltransferase catalyzes methyl‐transfer from S‐adenosyl‐L‐methionine to N7 atom of the semi‐conserved G46 base in tRNA. Aquifex aeolicus is a hyper thermophilic eubacterium that grows at close to 95°C. A. aeolicus tRNA (m7G46) methyltransferase [TrmB] has an elongated C‐terminal region as compared with mesophilic counterparts. In this study, the authors focused on the functions of this C‐terminal region. Analytic gel filtration chromatography and amino acid sequencing reveled that the start point (Glu202) of the C‐terminal region is often cleaved by proteases during purification steps and the C‐terminal region tightly binds to another subunit even in the presence of 6M urea. Because the C‐terminal region contains abundant basic amino acid residues, the authors assumed that some of these residues might be involved in tRNA binding. To address this idea, the authors prepared eight alanine substitution mutant proteins. However, measurements of initial velocities of these mutant proteins suggested that the basic amino acid residues in the C‐terminal region are not involved in tRNA binding. The authors investigated effects of the deletion of the C‐terminal region. Deletion mutant protein of the C‐terminal region (the core protein) was precipitated by incubation at 85°C, while the wild type protein was soluble at that temperature, demonstrating that the C‐terminal region contributes to the protein stability at high temperatures. The core protein had a methyl‐transfer activity to yeast tRNAPhe transcript. Furthermore, the core protein slowly methylated tRNA transcripts, which did not contain G46 base. Moreover, the modified base was identified as m7G by two‐dimensional thin layer chromatography. Thus, the deletion of the C‐terminal region causes nonspecific methylation of N7 atom of guanine base(s) in tRNA transcripts. Proteins 2008.

RNA recognition mechanism of eukaryote tRNA (m7G46) methyltransferase (Trm8–Trm82 complex)

Yeast tRNA (m7G46) methyltransferase contains two protein subunits (Trm8 and Trm82). To address the RNA recognition mechanism of the Trm8–Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNAPhe transcripts. Both the D‐stem and T‐stem structures were required for efficient methyl‐transfer. To clarify the role of the D‐stem structure, we tested four mutant transcripts, in which tertiary base pairs were disrupted. The tertiary base pairs were important but not essential for the methyl‐transfer to yeast tRNAPhe transcript, suggesting that these base pairs support the induced fit of the G46 base into the catalytic pocket.

Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNAMet (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus , an extreme-thermophilic eubacterium

Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus, in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (Δdus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Δdus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA(Phe) purified from the Δdus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures.