Hiroyuki Hori

Maturation of transfer RNA molecules often requires removal of intronic sequences by endonucleases that recognize diverse RNA secondary structures. Archaeal splicing endonucleases [versatile RNA-splicing endonucleases (VSENs)] exhibit remarkable substrate versatility, yet the structural basis for this broad specificity has remained unclear. Here, we report the 1.8-Å crystal structure of ARMAN-2, an ϵ2-type VSEN from Candidatus Micrarchaeum acidiphilum, in complex with a synthetic bulge–helix–bulge RNA. The structure reveals that a lineage-specific insertion, the ARMAN-specific loop (ASL), interacts with the bulged region of the RNA and helps to orient the scissile phosphate for catalysis via conserved tyrosine and lysine residues. Functional assays confirmed the essential role of the ASL in substrate binding and cleavage. Structural comparisons with (αβ)2-type Crenarchaeal VSENs, which contain a distinct Crenarchaea-specific loop (CSL), and with a eukaryotic equivalent, the TSEN complex, which harbors a previously uncharacterized eukaryotic-specific loop (ESL), uncovered mechanistic convergence across domains of life. We show that the ESL occupies a position analogous to the ASL and CSL, and likely supports bulge stabilization in long introns. These findings establish a mechanistic model for broad substrate recognition by VSENs and suggest that loop-mediated RNA positioning co-evolved with intron complexity in archaeal and eukaryotic lineages.

Methyltransferase ribozyme 1 (MTR1) is a catalytic RNA that has been isolated from a random RNA pool by in vitro selection. The ribozyme catalyzes site-specific formation of 1-methyl adenosine (m¹A) using 6-methyl guanine (m⁶G) as a methyl group donor. The ribozyme has been extensively characterized by biochemical and structural analyses. Here, we describe high-throughput screening of single point mutants in the catalytic domain of MTR1 and determine their effect on ribozyme activity. Our mutational profiling method successfully assessed the activity of the 141 MTR1 variants tested in each experiment and revealed that the ribozyme is very sensitive to nucleotide substitutions in the catalytic core domain. Our technique can be applied to methyltransferase ribozymes that catalyze formation of different modifications such as 7-methylguanosine (m⁷G) or 3-methylcytidine (m³C).

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR or DNA cloning. The oligonucleotide design, however, is a challenge to non-experts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the Rational design of Oligonucleotides, Calculated with Kinetic parameters for Enhanced in vitro Transcription (ROCKET). The Python tool employs thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2’-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA ^Ser_CGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNA^Ser, tRNA^Tyrand tRNA^Leu, the D-loop structure formed by interaction with the long variable region is important. For tRNA^Gln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase. In this review, I focus on the functions and structures of these tRNA modification enzymes and the modified nucleosides they produce. Biochemical, biophysical and structural studies of tRNA 4-thiouridine synthetase, tRNA methyltransferases and tRNA deaminase have established the concept that the THUMP domain captures the 3′-end of RNA (in the case of tRNA, the CCA-terminus). However, in some cases, this concept is not simply applicable given the modification patterns observed in tRNA. Furthermore, THUMP-related proteins are involved in the maturation of other RNAs as well as tRNA. Moreover, the modified nucleosides, which are produced by the THUMP-related tRNA modification enzymes, are involved in numerous biological phenomena, and the defects of genes for human THUMP-related proteins are implicated in genetic diseases. In this review, these biological phenomena are also introduced.

4-Thiouridine (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 thermophilustRNAs, which abundantly contain 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 aT. thermophilustRNA 4-thiouridine 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 inS. cerevisiaecells.