Language：English   Publisher：Cold Spring Harbor Laboratory  

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.

DOI： 10.1101/2023.02.27.530264

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).

DOI： 10.1016/bs.mie.2023.02.021

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

There is a multitude of small (<100nt) RNAs that serve diverse functional roles in biology. Key amongst these is transfer RNA (tRNA), which is among the most ancient RNAs and is part of the translational apparatus in every domain of life. Transfer RNAs are also the most heavily modified class of RNAs. They are essential and their misregulation, due to mutated sequences or loss of modification, can lead to disease. Because of the severe phenotypes associated with mitochondrial tRNA defects in particular, the desire to deliver repaired tRNAs via droplets such as lipid nanoparticles or other compartments is an active area of research. Here we describe how to use our tRNA Structure-seq method to study tRNAs and other small RNAs in two different biologically relevant contexts, peptide-rich droplets and in vivo.

DOI： 10.1016/bs.mie.2023.05.006

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

DOI： 10.1016/j.jbc.2022.102759

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4-Thiourdine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s4U in tRNA are ongoing in the tRNA modification field. s4U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s4U is essential for progress in the fields of RNA technologies and tRNA modification. Here we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s4U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation, however addition of a denaturation step improves the limit of detection. Thermus thermophilus tRNAs, which abundantly contains 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s4U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from a T. thermophilus tRNA 4-thiuridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s4U detection system is that MTSEA biotin-XX reacts only with s4U and not with other sulfur-containing modified nucleosides such as s2U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s4U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s4U detection system can also be used for the analysis of s4U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs in S. cerevisiae cells.

DOI： 10.1261/rna.079445.122

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RNA structure plays roles in myriad cellular events including transcription, translation, and RNA processing. Genome-wide analyses of RNA secondary structure in vivo by chemical probing have revealed critical structural features of mRNAs and long ncRNAs. Here, we examine the in vivo secondary structure of a small RNA class, tRNAs. Study of tRNA structure is challenging because tRNAs are heavily modified and strongly structured. We introduce "tRNA structure-seq," a new workflow that accurately determines in vivo secondary structures of tRNA. The workflow combines dimethyl sulfate (DMS) probing, ultra-processive RT, and mutational profiling (MaP), which provides mutations opposite DMS and natural modifications thereby allowing multiple modifications to be identified in a single read. We applied tRNA structure-seq to E. coli under control and stress conditions. A leading folding algorithm predicts E. coli tRNA structures with only ∼80% average accuracy from sequence alone. Strikingly, tRNA structure-seq, by providing experimental restraints, improves structure prediction under in vivo conditions to ∼95% accuracy, with more than 14 tRNAs predicted completely correctly. tRNA structure-seq also quantifies the relative levels of tRNAs and their natural modifications at single nucleotide resolution, as validated by LC-MS/MS. Our application of tRNA structure-seq yields insights into tRNA structure in living cells, revealing that it is not immutable but has dynamics, with partial unfolding of secondary and tertiary tRNA structure under heat stress that is correlated with a loss of tRNA abundance. This method is applicable to other small RNAs, including those with natural modifications and highly structured regions.

DOI： 10.1073/pnas.2201237119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites.

DOI： 10.3390/ijms23074046

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acs.biochem.1c00012

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Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols for using inverse folding software to design a ribozyme sequence that will fold to a known ribozyme secondary structure and for testing the catalytic activity of the sequence experimentally. This protocol is able to design sequences that include pseudoknots, which is important as all naturally occurring full-length ribozymes have pseudoknots. The starting point is the known pseudoknot-containing secondary structure of the ribozyme and knowledge of any nucleotides whose identity is required for function. The output of the protocol is a set of sequences that have been tested for function. Using this protocol, we were previously successful at designing highly active double-pseudoknotted HDV ribozymes.

DOI： 10.1007/978-1-0716-0716-9_8

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RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5' and 3' ends of the cloverleaf core, and these extensions get trimmed before addition of the 3'-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5' and 3' ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

DOI： 10.1073/pnas.1913418116

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg2+free. We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

DOI： 10.1021/acs.biochem.9b00578

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Design of RNA sequences that adopt functional folds establishes principles of RNA folding and applications in biotechnology. Inverse folding for RNAs, which allows computational design of sequences that adopt specific structures, can be utilized for unveiling RNA functions and developing genetic tools in synthetic biology. Although many algorithms for inverse RNA folding have been developed, the pseudoknot, which plays a key role in folding of ribozymes and riboswitches, is not addressed in most algorithms. For the few algorithms that attempt to predict pseudoknot-containing ribozymes, self-cleavage activity has not been tested. Herein, we design double-pseudoknot HDV ribozymes using an inverse RNA folding algorithm and test their kinetic mechanisms experimentally. More than 90% of the positively designed ribozymes possess self-cleaving activity, whereas more than 70% of negative control ribozymes, which are predicted to fold to the necessary structure but with low fidelity, do not possess it. Kinetic and mutation analyses reveal that these RNAs cleave site-specifically and with the same mechanism as the WT ribozyme. Most ribozymes react just 50- to 80-fold slower than the WT ribozyme, and this rate can be improved to near WT by modification of a junction. Thus, fast-cleaving functional ribozymes with multiple pseudoknots can be designed computationally.

DOI： 10.1093/nar/gky1118

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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.

DOI： 10.3390/microorganisms6040110

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DOI： 10.1093/jb/mvy037

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DOI： 10.1038/s41467-018-04415-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (Delta speB and Delta speD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (&gt;75 degrees C) in minimal medium, both strains survived at 80 degrees C when they were cultured at 70 degrees C until the mid-log phase and then shifted to 80 degrees C. We therefore prepared the Delta speB and Delta speD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N-7-methylguanosine at position 46, 5-methyluridine at position 54 and N-1-methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNA(Tyr), tRNA(His), rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

DOI： 10.1111/gtc.12502

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

TrmFO is a N-5, N-10-methylenetetrahydrofolate (CH2THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5)U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 degrees C). In T.thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5)U54 and TrmFO in the network, we constructed the trmFO gene disruptant (trmFO) strain of T.thermophilus. Although this strain did not show any growth retardation at 70 degrees C, it showed a slow-growth phenotype at 50 degrees C. Nucleoside analysis showed increase in 2-O-methylguanosine at position 18 and decrease in N-1-methyladenosine at position 58 in the tRNA mixture from the trmFO strain at 50 degrees C. These invivo results were reproduced by invitro experiments with purified enzymes. Thus, we concluded that the m(5)U54 modification have effects on the other modifications in tRNA through the network at 50 degrees C. S-35 incorporations into proteins showed that the protein synthesis activity of trmFO strain was inferior to the wild-type strain at 50 degrees C, suggesting that the growth delay at 50 degrees C was caused by the inferior protein synthesis activity.

DOI： 10.1111/gtc.12376

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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 (Delta dus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Delta dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) transcript at 60A degrees 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 Delta dus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures.

DOI： 10.1093/jb/mvv066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The solid-phase DNA probe method, in which a target transfer RNA (tRNA) is hybridized with a complementary DNA oligomer, is generally used for tRNA purification. However, purification of tRNAs from thermophiles by this method is not easy because of their high melting temperatures. To overcome this problem, the use of tetraalkylammonium salts was previously reported [Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89]. In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)(8) oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A(8) sequences was annealed. The probe sequence for tRNA purification was synthesized in column with Klenow enzyme. This DNA fixation method enabled us to use the column repeatedly and to wash the column with warmed buffers. Thus, this DNA fixation method is economical as compared with the previous method using the biotinylated DNA probe.

DOI： 10.1093/jb/mvu089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-L-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N-and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.

DOI： 10.1074/jbc.M113.485128

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The conserved U54 in tRNA is often modified to 5-methyluridine (m(5)U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m(5)U54 is produced by folate/FAD-dependent tRNA (m(5)U54) methyltransferase (TrmFO). TrmFO utilizes N-5,N-10-methylenetetrahydrofolate (CH2THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [C-14]CH2THF was supplied from [C-14]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 m(1)A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m(5)U54, m(1)A58, and s(2)U54 modifications on m(5)s(2)U54 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.

DOI： 10.1074/jbc.M112.390112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (alpha'(2), alpha(4) and alpha(2)beta(2)) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an epsilon(2) homodimer and has broad substrate specificity like the alpha(2)beta(2) type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the epsilon(2) homodimer of ARMAN-2 EndA. The structure reveals that the epsilon protomer is separated into three novel units (alpha(N), alpha and beta(C)) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of epsilon(2) and alpha(2)beta(2) EndAs were separately acquired through different evolutionary processes.

DOI： 10.1093/nar/gks826

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