Details of a Researcher

Papers - Kanai, Akio

Division display >> ／ All the affair displays 1 - 81 of about 81

Possible acquisition and molecular evolution of vpu genes inferred through comprehensive sequence analysis of Human and Simian Immunodeficiency Viruses

Naruki, M., Saito, M., Nomaguchi, M., and Kanai, A.

Journal of Molecular Evolution (Springer Nature) in press 2025.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Vpu, an accessory protein of human immunodeficiency virus-1 (HIV-1), plays a crucial role in viral particle production and significantly contributes to HIV virulence. However, the evolution of the vpu gene remains poorly understood. We conducted a computational analysis of approximately 39,000 simian immunodeficiency virus (SIV) and HIV sequences, focusing on 141 representative Vpu proteins. Phylogenetic analysis classified the SIV and HIV strains into four major types based on their Vpu proteins: Vpu-type 1 (ancestral, found in SIVs such as SIVmon and SIVgsn), Vpu-type 2 (SIVgor and HIV-1 group O), Vpu-type 3 (SIVcpz), and Vpu-type 4 (HIV-1 group M and N). Notably, Vpu-type 1 exhibited variability in gene length, genome length, and the overlap between vpu and env compared with other Vpu-types. A phylogenetic tree was constructed using 426 nucleotide sequences from HIV-1, HIV-2, and SIVs focusing on the region between the pol and env genes. Vpu-type 1 was closely clustered with SIVasc and SIVsyk, lacking both vpu and vpx. The similarities observed between vpu and genes such as vpr and env suggest that vpu originated within the SIV genome. In addition, a phylogenetic tree constructed from 252 Vpu-type 4a sequences from the HIV pandemic strain and 135 sequences of circulating recombinant forms of HIV-1 revealed 18 distinct protein subtypes, exceeding the number of previously recognized subtypes. The systematic analysis of the sequences from large datasets has enabled a detailed characterization of the transition states of vpu, enhancing our understanding of the processes driving viral diversity.

Access to Document (DOI)

An internal loop region is responsible for inherent target specificity of bacterial Cold-shock proteins

Hasegawa, S., Inose, R., Igarashi, M., Tsurumaki, M., Saito, M., Yanagisawa, T., Kanai, A., and Morita, T.

RNA (Cold Spring Harbor Laboratory Press) 31 ( 1 ) 67 - 85 2025.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 13558382

Cold shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold shock domain (CSD) that contains RNA binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD and the N-terminal portion with Lys4 of CspA are important for determining their target specificities. Pull-down assays suggest these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding of the functional specialization of Csps.

Access to Document (DOI)

Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences

Tsurumaki, M., Sato, A., Saito, M., and Kanai, A.

Journal of Bacteriology (American Society for Microbiology) 206 ( 7 ) e0007424 2024.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 00219193

ABSTRACT
The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of Candidatus (Ca.) Saccharibacteria and the 16S rRNA genes of Ca. Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI from Ca. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs.

IMPORTANCE
Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in most bacterial phyla. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.

Access to Document (DOI)

Microbiome analysis of the restricted bacteria in radioactive element-containing water at the Fukushima Daiichi Nuclear Power Station

Warashina T, Sato A, Hinai H, Shaikhutdinov N, Shagimardanova E, Mori H, Tamaki S, Saito M, Sanada Y, Sasaki Y, Shimada K, Dotsuta Y, Kitagaki T, Maruyama S, Gusev O, Narumi I, Kurokawa K, Morita T, Ebisuzaki T, Nishimura A, Koma Y, Kanai A.

Applied Enviromental Microbiology (American Society for Microbiology) 90 ( 4 ) e0211323 2024.04

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 00992240

A major incident occurred at the Fukushima Daiichi Nuclear Power Station following the tsunami triggered by the Tohoku-Pacific Ocean Earthquake in March 2011, whereby seawater entered the torus room in the basement of the reactor building. Here, we identify and analyze the bacterial communities in the torus room water and several environmental samples. Samples of the torus room water (1 × 10exp9 Bq 137Cs/L) were collected by the Tokyo Electric Power Company Holdings from two sampling points between 30 cm and 1 m from the bottom of the room (TW1) and the bottom layer (TW2). A structural analysis of the bacterial communities based on 16S rRNA amplicon sequencing revealed that the predominant bacterial genera in TW1 and TW2 were similar. TW1 primarily contained the genus Limnobacter, a thiosulfate-oxidizing bacterium. γ-Irradiation tests on Limnobacter thiooxidans, the most closely related phylogenetically found in TW1, indicated that its radiation resistance was similar to ordinary bacteria. TW2 predominantly contained the genus Brevirhabdus, a manganese-oxidizing bacterium. Although bacterial diversity in the torus room water was lower than seawater near Fukushima, ~70% of identified genera were associated with metal corrosion. Latent environment allocation-an analytical technique that estimates habitat distributions and co-detection analyses-revealed that the microbial communities in the torus room water originated from a distinct blend of natural marine microbial and artificial bacterial communities typical of biofilms, sludge, and wastewater. Understanding the specific bacteria linked to metal corrosion in damaged plants is important for advancing decommissioning efforts.

Access to Document (DOI)

Genome-resolved metaproteogenomic and nanosolid characterization of an inactive vent chimney densely colonized by enigmatic DPANN archaea

Takamiya H, Kouduka M, Kato S, Suga H, Oura M, Yokoyama T, Suzuki M, Mori M, Kanai A, Suzuki Y.

ISME J. (Oxford University Press) 18 ( 1 ) wrae207 2024.11

Research paper (scientific journal), Accepted, ISSN 17517362

Recent successes in the cultivation of DPANN archaea with their hosts have demonstrated an episymbiotic lifestyle, whereas the lifestyle of DPANN archaea in natural habitats is largely unknown. A free-living lifestyle is speculated in oxygen-deprived fluids circulated through rock media, where apparent hosts of DPANN archaea are lacking. Alternatively, DPANN archaea may be detached from their hosts and/or rock surfaces. To understand the ecology of rock-hosted DPANN archaea, rocks rather than fluids should be directly characterized. Here, we investigated a deep-sea hydrothermal vent chimney without fluid venting where our previous study revealed the high proportion of Pacearchaeota, one of the widespread and enigmatic lineages of DPANN archaea. Using spectroscopic methods with submicron soft X-ray and infrared beams, the microbial habitat was specified to be silica-filled pores in the inner chimney wall comprising chalcopyrite. Metagenomic analysis of the inner wall revealed the lack of biosynthetic genes for nucleotides, amino acids, cofactors, and lipids in the Pacearchaeota genomes. Genome-resolved metaproteomic analysis clarified the co-occurrence of a novel thermophilic lineage actively fixing carbon and nitrogen and thermophilic archaea in the inner chimney wall. We infer that the shift in metabolically active microbial populations from the thermophiles to the mesophilic DPANN archaea occurs after the termination of fluid venting. The infilling of mineral pores by hydrothermal silica deposition might be a preferred environmental factor for the colonization of free-living Pacearchaeota with ultrasmall cells depending on metabolites synthesized by the co-occurring thermophiles during fluid venting.

Access to Document (DOI)

Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei

Motofumi Saito, Rerina Inose, Asako Sato, Masaru Tomita, Haruo Suzuki & Akio Kanai

Journal of Molecular Evolution (Springer Nature) 91 ( 5 ) 669 - 686 2023.10

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 00222844

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5′ end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Access to Document (DOI)

Identification of attenuators of transcriptional termination: implications for RNA regulation in Escherichia coli.

Morita, T., Majdalani, N., Miura, M. C., Inose, R., Oshima, T., Tomita, M., Kanai, A., and Gottesman, S.

mBio (American Society for Microbiology ) 13 ( 6 ) e0237122 2022.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 21612129

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3′ portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling.

Access to Document (DOI)

Features of smaller ribosomes in Candidate Phyla Radiation (CPR) bacteria revealed with a molecular evolutionary analysis

Megumi Tsurumaki, Motofumi Saito, Masaru Tomita, and Akio Kanai

RNA (Cold Spring Harbor Laboratory Press) 28 ( 8 ) 1041 - 1057 2022.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 13558382

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100-150 amino acids, whereas the position of the peak varies in the range of 100-300 amino acids in free-living non-CPR bacteria, and at around 100-200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

Access to Document (DOI)

Expansion of non-canonical V-arm-containing tRNAs in eukaryotes.

Hamashima, K., Tomita, M. and Kanai, A.

Molecular Biology and Evolution (Oxford Academic) 33 ( 2 ) 530 - 540 2016

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Transfer RNA (tRNA) is essential for the translation of genetic information into proteins, and understanding its molecular evolution is important if we are to understand the genetic code. In general, long variable-arm (V-arm) structures form in tRNALeu, tRNASer, and bacterial and organellar tRNATyr. However, as we have previously reported, noncanonical V-arms occur in nematode tRNAGly and tRNAIle, and potentially affect translational fidelity. Here, we comprehensively analyzed 69 eukaryotic genome sequences and examined the evolutionary divergence of the V-arm-containing tRNAs. In total, 253 V-arm-containing tRNAs, with neither leucine nor serine anticodons, were identified in organisms ranging from nematodes to fungi, plants, and vertebrates. We defined them as “noncanonical V-arm-containing tRNAs” (nov-tRNAs). Moreover, 2,415 nov-tRNA-like sequences lacking some of the conserved features of tRNAs were also identified, largely in vertebrate genomes. These nov-tRNA/nov-tRNA-like sequences can be categorized into three types, based on differences in their possible evolutionary origins. The type A nov-tRNAs in nematodes probably evolved not only from tRNALeu but also from tRNASer and other isotypes on several independent occasions. The type B nov-tRNAs are dispersed abundantly throughout vertebrate genomes, and seem to have originated from retrotransposable elements. The type C nov-tRNAs may have been acquired from plant chloroplasts or from bacteria through horizontal transfer. Our findings provide unexpected insight into the evolution of the tRNA molecule, which was diverse and occurred independently in nematodes, vertebrates, and plants.

Access to Document (DOI)

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth

Noro, E., Mori, M., Makino, G., Takai, Y., Ohnuma, S., Sato, A., Tomita, M., Nakahigashi, K. and Kanai, A.

RNA Biology (Taylor & Francis Group) 14 ( 2 ) 206 - 218 2017

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Access to Document (DOI)

Identification, expression, and molecular evolution of microRNAs in the “living fossil” Triops cancriformis (tadpole shrimp).

Ikeda T. K., Hirose, Y., Hiraoka, K., Noro, E., Fujishima, K., Tomita, M. and Kanai, A.

RNA (Cold Spring Harbor Press) 21 ( 2 ) 230 - 242 2015.01

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a “living fossil,” whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first–fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1–3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a “living fossil.” However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.

Access to Document (DOI)

Tri-split tRNA is a transfer RNA made from 3 transcripts that provides insight into the evolution of fragmented tRNAs in archaea.

Fujishima, K., Sugahara, J., Kikuta, K., Hirano, R., Sato, A., Tomita, M. and Kanai, A.

Proceedings of the National Academy of Sciences U. S. A. (National Academy of Sciences) 106 ( 8 ) 2683 - 2687 2009.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNAGly's with synonymous codons are created from 1 constitutive 3′ half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a “tri-split tRNA.” Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in intron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs.

Access to Document (DOI)

Nematode-specific tRNAs that decode an alternative genetic code for leucine

Hamashima, K., Fujishima, K., Masuda, T., Sugahara, J., Tomita, M. and Kanai, A.

Nucleic Acids Research (Oxford Academic) 40 ( 8 ) 3653 - 3662 2012.04

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Class II transfer RNAs (tRNAs), including tRNA(Leu) and tRNA(Ser), have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNA(Leu). In vitro aminoacylation assays showed that nev-tRNA(Gly) and nev-tRNA(Ile) are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA(Gly) decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.

Access to Document (DOI)

A novel three-unit tRNA splicing endonuclease found in ultrasmall archaea possesses broad substrate specificity.

Fujishima, K., Sugahara, J., Miller, CS., Baker BJ., Di Giulio, M., Takesue, K., Sato, A., Tomita, M., Banfield, JF. and Kanai, A.

Nucleic Acids Research (Oxford Academic) 39 ( 22 ) 9695 - 9704 2011

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α 4 , homodimeric: α 2 , and heterotetrameric: (αβ) 2 ] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε 2 ) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε 2 endonuclease cleaves both canonical and non-canonical bulge–helix–bulge motifs, similar to that of (αβ) 2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ) 2 endonuclease. Thus, the discovery of ε 2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.

Access to Document (DOI)

Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus.

Kanai, A., Sato, A., Fukuda, Y. Okada, K., Matsuda, T., Sakamoto, T., Muto, Y., Yokoyama, S., Kawai, G. and Tomita, M.

RNA (Cold Spring Harbor Laboratory Press) 15 ( 3 ) 420-431 2009.01

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein.

Access to Document (DOI)

Homo-trimeric structure of the ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus

Gota Kawai, Kiyoshi Okada, Seiki Baba, Asako Sato, Taiichi Sakamoto and Akio Kanai

The Journal of Biochemistry (Oxford Academic) 175 ( 6 ) 671 - 676 2024.06

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 0021924X

Crystal structure of a ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains, N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by PAGE and NMR. Interestingly, well dispersed NMR signals were observed at 318 K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and RNase E show a structural similarity in their RNA binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain.

Access to Document (DOI)

Distinct Expansion of Group II Introns During Evolution of Prokaryotes and Possible Factors Involved in Its Regulation

Masahiro C. Miura, Shohei Nagata, Satoshi Tamaki, Masaru Tomita, and Akio Kanai

Frontiers in Microbiology (Frontiers Media SA) 13 849080 2022.02

Research paper (scientific journal), Last author, Corresponding author, Accepted

Group II introns (G2Is) are ribozymes that have retroelement characteristics in prokaryotes. Although G2Is are suggested to have been an important evolutionary factor in the prokaryote-to-eukaryote transition, comprehensive analyses of these introns among the tens of thousands of prokaryotic genomes currently available are still limited. Here, we developed a bioinformatic pipeline that systematically collects G2Is and applied it to prokaryotic genomes. We found that in bacteria, 25% (447 of 1,790) of the total representative genomes had an average of 5.3 G2Is, and in archaea, 9% (28 of 296) of the total representative genomes had an average of 3.0 G2Is. The greatest number of G2Is per genome was 101 in Arthrospira platensis (phylum Cyanobacteriota). A comprehensive sequence analysis of the intron-encoded protein (IEP) in each G2I sequence was conducted and resulted in the addition of three new IEP classes (U1–U3) to the previous classification. This analysis suggested that about 30% of all IEPs are non-canonical IEPs. The number of G2Is per genome was defined almost at the phylum level, and at least in the following two phyla, Firmicutes, and Cyanobacteriota, the type of IEP was largely associated as a factor in the G2I increase, i.e., there was an explosive increase in G2Is with bacterial C-type IEPs, mainly in the phylum Firmicutes, and in G2Is with CL-type IEPs, mainly in the phylum Cyanobacteriota. We also systematically analyzed the relationship between genomic signatures and the mechanism of these increases in G2Is. This is the first study to systematically characterize G2Is in the prokaryotic phylogenies.

Access to Document (DOI)

Dataset for proteomic analysis of Escherichia coli before and after severe heat shock stress

Huang, M., Miura, M. C., Inose, R., Sato, A., Kamezaki, K., Tomita, M., Mori, M., Arakawa, K., and Kanai, A.

Journal of Proteome Data and Methods (Japanese Proteomics Society, JPrOS) 4 1 - 3 2022

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Access to Document (DOI)

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation

Hayashi, Y., Mori, M., Igarashi, K., Tanaka, K., Takehara, A., Ito-Matsuoka, Y., Kanai, A., Yaegasgi, N., Soga, T. and Matsui, Y.

Biology of Reproduction (Oxford University Press) 103 ( 4 ) 717 - 735 2020.07

Research paper (scientific journal), Accepted, ISSN 00063363

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Access to Document (DOI)

Behavioral and brain- transcriptomic synchronization between the two opponents of a fighting pair of the fish Betta splendens.

Vu, T.-D., Iwasaki, Y., Shigenobu, S., Maruko, A., Oshima, K., Iioka E., Huang, C.-L., Abe, T., Tamaki, S., Lin, Y.-W., Chen, C.-K., Lu, M.-Y., Hojo, M., Wang, H.-V., Tzeng, S.-F., Huang, H.-J., Kanai, A., Gojobori, T., Chiang, T.-Y., Sun, H. S., Li, W.-H., and Okada, N.

PLOS Genetics (PLOS) 16 ( 6 ) 2020.06

Research paper (scientific journal), Accepted, ISSN 15537390

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.

Access to Document (DOI)

Editorial: Current Advances in the Research of RNA Regulatory Enzymes

Kanai, A. and Yoshihisa, T.

Frontiers in Genetics (Frontiers) 10 ( 973 ) 2019.10

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Large-scale Molecular Evolutionary Analysis Uncovers a Variety of Polynucleotide Kinase Clp1 Family Proteins in the Three Domains of Life

Saito, M., Sato, A., Nagata, S., Tamaki, S., Tomita, M., Suzuki, H., and Kanai, A.

Genome Biology and Evolution (Genome Biology and Evolution) 11 ( 10 ) 2713 - 2726 2019.09

Research paper (scientific journal), Last author, Corresponding author, Accepted

© 2019 The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. Clp1, a polyribonucleotide 5′-hydroxyl kinase in eukaryotes, is involved in pretRNA splicing and mRNA 3′-end formation. Enzymes similar in amino acid sequence to Clp1, Nol9, and Grc3, are present in some eukaryotes and are involved in prerRNA processing. However, our knowledge of how these Clp1 family proteins evolved and diversified is limited. We conducted a large-scale molecular evolutionary analysis of the Clp1 family proteins in all living organisms for which protein sequences are available in public databases. The phylogenetic distribution and frequencies of the Clp1 family proteins were investigated in complete genomes of Bacteria, Archaea and Eukarya. In total, 3,557 Clp1 family proteins were detected in the three domains of life, Bacteria, Archaea, and Eukarya. Many were from Archaea and Eukarya, but a few were found in restricted, phylogenetically diverse bacterial species. The domain structures of the Clp1 family proteins also differed among the three domains of life. Although the proteins were, on average, 555 amino acids long (range, 196-2,728), 122 large proteins with >1,000 amino acids were detected in eukaryotes. These novel proteins contain the conserved Clp1 polynucleotide kinase domain and various other functional domains. Of these proteins, >80% were from Fungi or Protostomia. The polyribonucleotide kinase activity of Thermus scotoductus Clp1 (Ts-Clp1) was characterized experimentally. Ts-Clp1 preferentially phosphorylates single-stranded RNA oligonucleotides (Km value for ATP, 2.5 μM), or single-stranded DNA at higher enzyme concentrations. We propose a comprehensive assessment of the diversification of the Clp1 family proteins and the molecular evolution of their functional domains.

Access to Document (DOI)

Systematic analysis of the binding surfaces between tRNAs and their respective aminoacyl tRNA synthetase based on structural and evolutionary data

Tamaki, S., Tomita, M., Suzuki, H. and Kanai, A.

Frontiers in Genetics (Frontiers in Genetics) 8 ( 227 ) 2018.01

Research paper (scientific journal), Last author, Corresponding author, Accepted

© 2018 Tamaki, Tomita, Suzuki and Kanai. To determine the mechanism underlying the flow of genetic information, it is important to understand the relationship between a tRNA and its binding enzyme, a member of the aminoacyl-tRNA synthetase (aaRS) family. We have developed a novel method to project the interacting regions of tRNA-aaRS complexes, obtained from their three-dimensional structures, onto two-dimensional space. The interacting surface between each tRNA and its aaRS was successfully identified by determining these interactions with an atomic distance threshold of 3.3 Å. We analyzed their interactions, using 60 mainly bacterial and eukaryotic tRNA-aaRS complexes, and showed that the tRNA sequence regions that interacted most strongly with each aaRS are the anticodon loop and the CCA terminal region, followed by the D-stem. A sequence conservation analysis of the canonical tRNAs was conducted in 83 bacterial, 182 archaeal, and 150 eukaryotic species. Our results show that the three tRNA regions that interact with the aaRS and two additional loop regions (D-loop and TψC-loop) known to be important for formation of the tRNA L-shaped structure are broadly conserved. We also found sequence conservations near the tRNA discriminator in the Bacteria and Archaea, and an enormous number of noncanonical tRNAs in the Eukaryotes. This is the first global view of tRNA evolution based on its structure and an unprecedented number of sequence data.

Access to Document (DOI)

Evolutionary analysis of HIV-1 Pol proteins reveals representative residues for viral subtype differentiation

Nagata, S., Imai, J., Makino, G., Tomita, M. and Kanai, A.

Frontiers in Microbiology 8 ( 2151 ) 2017.11

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

An archaeal RNA binding protein, FAU-1, is a novel ribonuclease related to rRNA stability in Pyrococcus and Thermococcus.

Ikeda, Y., Okada, Y., Sato, A., Kanai, T., Tomita, M., Atomi, H. and Kanai, A.

Scientific Reports 7 12674 2017.10

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells.

Hayashi, Y., Otsuka, K., Ebina, M., Igarashi, K., Takehara, A., Matsumoto, M., Kanai, A., Igarashi, K., Soga, T. and Matsui, Y.

Proceedings of the National Academy of Sciences. U. S. A. 114 ( 31 ) 8289 - 8294 2017.08

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Involvement of nucleotide diphosphate kinase 2 in the reopening of the sensitive period of filial imprinting of domestic chicks (Gallus gallus domesticus).

Yamaguchi, S., Aoki, N., Takehara, A., Mori, M., Kanai, A., Matsushima, T. and Homma, K. J.

Neuroscience Letters (Elsevier) 612 32 - 37 2016

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp).

Hirose, Y., Ikeda, T. K., Noro, E., Hiraoka, K., Tomita, M. and Kanai, A.

BMC Genetics (BioMed Central) 16 ( 83 ) 2015.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Background
In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNAGly(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st − 4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences.

Results
We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRFSer(GCU)s were abundantly produced from the 5’ half regions of the parental tRNA, whereas mitochondrial tRFVal(UAC)s were mainly produced from the 3’ end regions. Highly abundant nuclear tRFs, tRFGly(GCC)s, tRFGly(CCC)s, tRFGlu(CUC)s, and tRFLys(CUU)s were derived from the 5’ half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development.

Conclusions
Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

Access to Document (DOI)

Disrupted tRNA genes and tRNA fragments: a perspective on tRNA gene evolution.

Kanai, A.

Life (Basel) 5 ( 1 ) 321 - 331 2015.01

Research paper (scientific journal), Single Work, Accepted

Access to Document (DOI)

Analysis of genetic code ambiguity arising from nematode-specific misacylated tRNAs.

Hamashima, K., Mori, M., Andachi, Y., Tomita, M., Kohara, Y. and Kanai, A.

PLOS ONE 10 ( 1 ) e0116981 2015.01

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Effect of codon adaptation on codon-level and gene-level translation efficiency in vivo.

Nakahigashi, K., Takai, Y., Shiwa, Y., Wada, M., Honma, M., Yoshikawa, H., Tomita, M., Kanai, A. and Mori. H.

BMC Genomics 15 ( 1 ) 1115 2014.12

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Welcome to the new tRNA world!

Akio Kanai

Frontiers in Genetics 5 336 2014.09

Research paper (scientific journal), Single Work, Accepted

Access to Document (DOI)

tRNA gene diversity in the three domains of life

Fujishima, K. and Kanai, A.

Frontiers in Genetics 5 142 2014.04

Research paper (scientific journal), Joint Work, Accepted

Access to Document (DOI)

Identification of highly-disrupted tRNA genes in nuclear genome of the red alga, Cyanidioschyzon merolae 10D

Soma, A., Sugahara, J., Onodera, A., Yachie, N., Kanai, A., Watanabe, S., Yoshikawa, H., Ohnuma, M., Kuroiwa, H., Kuroiwa, T. and Sekine, Y.

Scientific Reports 3 2321 2013.07

Research paper (scientific journal), Joint Work, Accepted

Comprehensive computational analysis of bacterial CRP/FNR superfamily and its target motifs reveals stepwise evolution of transcriptional networks.

Matsui, M., Tomita, M. and Kanai, A.

Genome Biology and Evolution (Oxford Academic) 5 ( 2 ) 267 - 282 2013.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

The cAMP receptor protein (CRP)/fumarate and nitrate reduction regulatory protein (FNR)-type transcription factors (TFs) are members of a well-characterized global TF family in bacteria and have two conserved domains: the N-terminal ligand-binding domain for small molecules (e.g., cAMP, NO, or O2) and the C-terminal DNA-binding domain. Although the CRP/FNR-type TFs recognize very similar consensus DNA target sequences, they can regulate different sets of genes in response to environmental signals. To clarify the evolution of the CRP/FNR-type TFs throughout the bacterial kingdom, we undertook a comprehensive computational analysis of a large number of annotated CRP/FNR-type TFs and the corresponding bacterial genomes. Based on the amino acid sequence similarities among 1,455 annotated CRP/FNR-type TFs, spectral clustering classified the TFs into 12 representative groups, and stepwise clustering allowed us to propose a possible process of protein evolution. Although each cluster mainly consists of functionally distinct members (e.g., CRP, NTC, FNR-like protein, and FixK), FNR-related TFs are found in several groups and are distributed in a wide range of bacterial phyla in the sequence similarity network. This result suggests that the CRP/FNR-type TFs originated from an ancestral FNR protein, involved in nitrogen fixation. Furthermore, a phylogenetic profiling analysis showed that combinations of TFs and their target genes have fluctuated dynamically during bacterial evolution. A genome-wide analysis of TF-binding sites also suggested that the diversity of the transcriptional regulatory system was derived by the stepwise adaptation of TF-binding sites to the evolution of TFs.

Access to Document (DOI)

Alternative genetic code for amino acids and transfer RNA revisited

Hamashima, K. and Kanai, A.

Biomolecular Concepts 4 ( 3 ) 309-318 2013

Research paper (scientific journal), Joint Work, Accepted

Genomic heterogeneity in a natural archaeal population suggests a model of tRNA gene disruption

Sugahara, J., Fujishima, K., Nunoura, T., Takaki, Y., Takami, H., Takai, K., Tomita, M. and Kanai, A.

PLoS ONE 7 ( 3 ) e32504 2012.03

Research paper (scientific journal), Joint Work, Accepted

Metatranscriptomic analysis of microbes in an ocean-front deep subsurface hot spring reveals novel small RNAs and type-specific tRNA degradation

Murakami, S., Fujishima, K., Tomita, M. and Kanai, A.

Applied and Environmental Microbiology (American Society for Microbiology) 78 ( 4 ) 1015 - 1022 2012.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000 m in Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected.

Access to Document (DOI)

X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: Insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity.

Hirata, A., Fujishima, K., Yamagami, R., Kawamura, T., Banfield, JF., Kanai, A. and Hori, H.

Nucleic Acids Research 40 ( 20 ) 10554-10566 2012

Research paper (scientific journal), Joint Work, Accepted

GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus

Sato, A., Soga, T., Igarashi, K., Takesue, K., Tomita, M. and Kanai, A.

Genes to Cells 16 1190-1199 2011.12

Research paper (scientific journal), Joint Work, Accepted

A screening system for artificial small RNAs that inhibit the growth of Escherichia coli

Mizuki Komasa, Kosuke Fujishima, Kiriko Hiraoka, Atsuko Shinhara, Baek-Seok Lee, Masaru Tomita and Akio Kanai

The Journal of Biochemistry 150 ( 3 ) 289-294 2011.09

Research paper (scientific journal), Joint Work, Accepted

Vertebrate-virus-encoded microRNAs and their sequence conservation

Takane, K. and Kanai, A.

Japanese Journal of Infectious Diseases 64 ( 5 ) 357-366 2011.09

Research paper (scientific journal), Joint Work, Accepted

Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli.

Shinhara, A., Matsui, M., Hiraoka, K., Nomura, W., Hirano, R., Nakahigashi, K., Tomita, M., Mori, H. and Kanai, A.

BMC Genomics (BioMed Central) 12 428 2011.08

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Background
In Escherichia coli, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of E. coli with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.

Results
We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three cis-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.

Conclusions
This comprehensive screen for E. coli sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the E. coli genome. We constructed the Escherichia coli Small RNA Browser (ECSBrowser; http://rna.iab.keio.ac.jp/), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study.

Access to Document (DOI)

Systems biology reveals microRNA-mediated gene regulation

Watanabe, Y. and Kanai, A.

Frontiers in Genetics 2 29 2011.06

Research paper (scientific journal), Joint Work, Accepted

tRNADB-CE 2011: tRNA gene database curated manually by experts.

Abe, T., Ikemura, T., Sugahara, J., Kanai, A., Ohara, Y., Uehara, H., Kinouchi, M., Kanaya, S., Yamada, Y., Muto, A. and Inokuchi, H.

Nucleic Acids Research 39 D210-D213 2011

Research paper (scientific journal), Joint Work, Accepted

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

Takuro Nunoura, Yoshihiro Takaki, Jungo Kakuta, Shinro Nishi, Junichi Sugahara, Hiromi Kazama, Gab-Joo Chee, Masahira Hattori, Akio Kanai, Haruyuki Atomi, Ken Takai and Hideto Takami

Nucleic Acids Research 39 ( 8 ) 3204-3223 2011

Research paper (scientific journal), Joint Work, Accepted

Comprehensive expressional analyses of antisense transcripts in colon cancer tissues using artificial antisense probes

Rintaro Saito, Keisuke Kohno, Yuki Okada, Yuko Osada, Koji Numata, Chihiro Kohama, Kazufumi Watanabe, Hajime Nakaoka, Naoyuki Yamamoto, Akio Kanai, Hiroshi Yasue, Soichiro Murata, Kuniya Abe, Masaru Tomita, Nobuhiro Ohkohchi, Hidenori Kiyosawa

BMC Medical Genomics 4 42 2011

Research paper (scientific journal), Joint Work

Large-scale tRNA intron transposition in the archaeal order Thermoproteales represents a novel mechanism of intron gain

Fujishima, K., Sugahara, J., Tomita, M. and Kanai, A.

Molecular Biology and Evolution (Oxford Academic) 27 ( 10 ) 2233 - 2243 2010.10

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Recently, diverse arrangements of transfer RNA (tRNA) genes have been found in the domain Archaea, in which the tRNA is interrupted by a maximum of three introns or is even fragmented into two or three genes. Whereas most of the eukaryotic tRNA introns are inserted strictly at the canonical nucleotide position (37/38), archaeal intron-containing tRNAs have a wide diversity of small tRNA introns, which differ in their numbers and locations. This feature is especially pronounced in the archaeal order Thermoproteales. In this study, we performed a comprehensive sequence comparison of 286 tRNA introns and their genes in seven Thermoproteales species to clarify how these introns have emerged and diversified during tRNA gene evolution. We identified 46 intron groups containing sets of highly similar sequences (>70%) and showed that 16 of them contain sequences from evolutionarily distinct tRNA genes. The phylogeny of these 16 intron groups indicates that transposition events have occurred at least seven times throughout the evolution of Thermoproteales. These findings suggest that frequent intron transposition occurs among the tRNA genes of Thermoproteales. Further computational analysis revealed limited insertion positions and corresponding amino acid types of tRNA genes. This has arisen because the bulge–helix–bulge splicing motif is required at the newly transposed position if the pre-tRNA is to be correctly processed. These results clearly demonstrate a newly identified mechanism that facilitates the late gain of short introns at various noncanonical positions in archaeal tRNAs.

Access to Document (DOI)

Transcription-associated mutagenesis increase protein sequence diversity more effectively than does random mutagenesis in Escherichia coli

Kim, H., Lee, B.-S., Tomita, M. and Kanai, A.

PLoS ONE 5 ( 5 ) e10567 2010.05

Research paper (scientific journal), Joint Work, Accepted

Permuted tRNA genes in the nuclear and nucleomorph genomes of photosynthetic eukaryotes.

Maruyama, S., Sugahara, J., Kanai, A. and Nozaki, H.

Molecular Biology and Evolution 27 ( 5 ) 1070-1076 2010.05

Research paper (scientific journal), Joint Work, Accepted

Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

Takane, K., Fujishima, K., Watanabe, Y., Sato, A., Saito, N., Tomita, M. and Kanai, A.

BMC Genomics 11 ( 101 ) 2010.02

Research paper (scientific journal), Joint Work, Accepted

Characterization of RNase HII substrate recognition using RNase HII-Argonaute chimeric enzymes from Pyrococcus furiosus

Sayaka Kitamura, Kosuke Fujishima, Asako Sato, Daisuke Tsuchiya, Masaru Tomita and Akio Kanai

Biochemical Journal 426 ( 3 ) 337-344 2010.01

Research paper (scientific journal), Joint Work, Accepted

Disrupted tRNA gene diversity and possible evolutionary scenarios

Sugahara, J., Fujishima, K., Morita, K., Tomita, M. and Kanai, A.

Journal of Molecular Evolution 69 ( 5 ) 497-504 2009.10

Research paper (scientific journal), Joint Work, Accepted

Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

Numata, K., Osada, Y., Okada, Y., Saito, R., Hiraiwa, N., Nakaoka, H., Yamamoto, N., Watanabe, K., Okubo, K., Kohama, C., Kanai, A., Abe, K. and Kiyosawa, H.

BMC Genomics 10 392 2009.08

Research paper (scientific journal), Joint Work, Accepted

Comprehensive analysis of archaeal tRNA genes reveals rapid increase of tRNA introns in the order Thermoproteales

Sugahara, J., Kikuta, K., Fujishima, K., Yachie, N., Tomita, M. and Kanai, A.

Molecular Biology and Evolution 25 ( 12 ) 2709-2716 2008.10

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Sequence evidence in the archaeal genome that tRNAs emerged through the combination of ancestral genes as 5’ and 3’ tRNA halves

Fujishima, K., Sugahara, J., Tomita, M. and Kanai, A.

PLoS ONE 3 ( 2 ) e1622 2008.02

Research paper (scientific journal), Joint Work, Accepted

Comparative expression analysis uncovers novel features of endogenous antisense transcription

Okada Y, Tashiro C, Numata K, Watanabe K, Nakaoka H, Yamamoto N, Okubo K, Ikeda R, Saito R, Kanai A, Abe K, Tomita M, Kiyosawa H.

Human Molecular Genetics 17 ( 11 ) 1631-1640 2008.02

Research paper (scientific journal), Joint Work, Accepted

Perspective in the evolution of human microRNAs: copy number expansion and acquisition of target gene specialization

Watanabe, Y., Tomita, M. and Kanai, A

Progress of Theoretical Physics Supplement 173 219-228 2008

Research paper (scientific journal), Joint Work, Accepted

Comparison and characterization of proteomes in the three domains of life using 2D correlation analysis

Fujishima, K., Komasa, M., Kitamura, S., Tomita, M. and Kanai, A

Progress of Theoretical Physics Supplement 173 206-218 2008

Research paper (scientific journal), Joint Work, Accepted

A novel processing pathway via a circular RNA intermediate of permutated tRNA genes in Cyanidioschyzon merolae

Soma, A., Onodera, A., Sugahara, J., Kanai, A., Yachie, N., Tomita, M., Kawamura, F. and Sekine, Y.

Science 318 450-453 2007.10

Research paper (scientific journal), Joint Work, Accepted

Computational analysis of microRNA-mediated antiviral defense in Humans.

Watanabe, Y., Kishi, A., Yachie, N., Kanai, A. and Tomita, M.

FEBS Letters 581 ( 24 ) 4603-4610 2007.10

Research paper (scientific journal), Joint Work, Accepted

Evolution of ribonuclease H genes in prokaryotes to avoid inheritance of redundant genes.

Kochiwa, H., Tomita, M. and Kanai, A.

BMC Evolutionary Biology 7 ( 128 ) 2007.07

Research paper (scientific journal), Joint Work, Accepted

Proteome-wide prediction of novel RNA/DNA-binding proteins using amino acid composition and periodicity in hyperthermophilic archeaon Pyrococcus furiosus.

Fujishima, K., Komasa, M., Kitamura, S., Suzuki, H., Tomita, M. and Kanai, A.

DNA Research 14 91-102 2007.06

Research paper (scientific journal), Joint Work

Comparative analysis of cis-encoded antisense RNAs in eukaryotes.

Numata, K., Okada, Y., Saito, R., Kiyosawa, H., Kanai, A., and Tomita, M.

Gene 392 134-141 2007.05

Research paper (scientific journal), Joint Work, Accepted

Multiple high throughput analyses monitor the response of E. coli to perturbations.

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Kakazu, Y., Sugawara, K., Igarashi, S., Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, Y., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H. and Tomita, M.

Science (AAAS) 316 593 - 597 2007.04

Research paper (scientific journal), Joint Work, Lead author, Accepted

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

Access to Document (DOI)

Computational method for microRNA target prediction.

Watanabe, Y., Tomita, M. and Kanai, A.

Methods in Enzymology 427 65-86. 2007

Research paper (scientific journal), Joint Work

Importance of systematic identification of RNA-binding proteins in a hyperthermophilic archaeon

KANAI AKIO

Viva Origino 34 144-147. 2006.12

Research paper (scientific journal), Single Work, Accepted

1H, 13C and 15N resonance assignments of the 2’-5’ RNA ligase-like protein from Pyrococcus furiosus.

Okada, K., Matsuda, T., Sakamoto, T., Muto, Y., Yokoyama, S., Kanai, A. and Kawai, G.

Journal of Biomolecular NMR 2006.02

Research paper (scientific journal), Joint Work, Accepted

Computational analysis of micro RNA targets in Caenorhabditis elegans.

Watanabe, Y., Yachie, N., Numata, K., Saito, R., Kanai, A. and Tomita, M.

Gene 365 2-10 2006.01

Research paper (scientific journal), Joint Work, Accepted

Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.

KANAI AKIO, Sato, A., Imoto, J. and Tomita, M.

Biochemical Journal 393 373-379 2006.01

Research paper (scientific journal), Joint Work, Accepted

Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificity and bivalent cation requirements.

Kochiwa, H., Itaya, M., Tomita, M. and KANAI A.

FEBS Journal 273 420-429 2006.01

Research paper (scientific journal), Joint Work, Accepted

SPLITS: a new program for predicting split and intron-containing tRNA genes at the genome level.

Sugahara, J., Yachie, N., Sekine, Y., Soma, A., Matsui, M., Tomita, M. and Kanai, A.

In Silico Biology 6 411-418 2006

Research paper (scientific journal), Joint Work, Accepted

Prediction of non-coding and antisense RNA genes in the Escherichia coli with Gapped Markov Model.

Yachie, N., Numata, K., Saito, R., Kanai, A. and Tomita, M.

Gene 372 171-181 2006

Research paper (scientific journal), Joint Work, Accepted

Solution structure of a GAAG tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus.

Okada, K., Takahashi, M., Sakamoto, T., Kawai, G., Nakamura, K. and Kanai, A.

Nucleosides, Nucleotides & Nucleic Acids 25 383-395 2006

Research paper (scientific journal), Joint Work, Accepted

Identification and expression analysis of mRNA-like non-coding RNAs in Drosophila.

Inagaki, S., Numata, K., Kondo, T., Tomita, M., Yasuda, K., Kanai, A. and Kageyama, Y.

Genes to Cells 10 1163-1173 2005.12

Research paper (scientific journal), Joint Work, Accepted

Toward large-scale modeling of the microbial cell for computer simulation

ISHII N, ROBERT M, NAKAYAMA Y, KANAI A, TOMITA M

Journal of Biotechnology 113 ( 1/3 ) 281-294 2004.09

Research paper (scientific journal), Joint Work, ISSN 01681656

Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus

Sato A, Kanai A, Itaya M, Tomita M

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 309 ( 1 ) 247-252 2003.09

Research paper (scientific journal), Joint Work, ISSN 0006-291X

Computational analysis of stop codon readthrough in D-melanogaster

Sato M, Umeki H, Saito R, Kanai A, Tomita M

BIOINFORMATICS 19 ( 11 ) 1371-1380 2003.07

Research paper (scientific journal), Joint Work, ISSN 1367-4803

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection

Numata K, Kanai A, Saito R, Kondo S, Adachi J, Wilming LG, Hume DA, Hayashizaki Y, Tomita M

GENOME RESEARCH 13 ( 6B ) 1301-1306 2003.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 1088-9051

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Access to Document (DOI)

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus

Kanai A, Oida H, Matsuura N, Doi H

BIOCHEMICAL JOURNAL 372 253-261 2003.05

Research paper (scientific journal), Joint Work, ISSN 0264-6021

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Okazaki Y, Furuno M, Kasukawa T, Adachi J, Bono H, Kondo S, Nikaido I, Osato N, Saito R, Suzuki H, Yamanaka I, Kiyosawa H, Yagi K, Tomaru Y, Hasegawa Y, Nogami A, Schonbach C, Gojobori T, Baldarelli R, Hill DP, Bult C, Hume DA, Quackenbush J, Schriml LM, Kanapin A, Matsuda H, Batalov S, Beisel KW, Blake JA, Bradt D, Brusic V, Chothia C, Corbani LE, Cousins S, Dalla E, Dragani TA, Fletcher CF, Forrest A, Frazer KS, Gaasterland T, Gariboldi M, Gissi C, Godzik A, Gough J, Grimmond S, Gustincich S, Hirokawa N, Jackson IJ, Jarvis ED, Kanai A, Kawaji H, Kawasawa Y, Kedzierski RM, King BL, Konagaya A, Kurochkin IV, Lee Y, Lenhard B, Lyons PA, Maglott DR, Maltais L, Marchionni L, McKenzie L, Miki H, Nagashima T, Numata K, Okido T, Pavan WJ, Pertea G, Pesole G, Petrovsky N, Pillai R, Pontius JU, Qi D, Ramachandran S, Ravasi T, Reed JC, Reed DJ, Reid J, Ring BZ, Ringwald M, Sandelin A, Schneider C, Semple CAM, Setou M, Shimada K, Sultana R, Takenaka Y, Taylor MS, Teasdale RD, Tomita M, Verardo R

NATURE 420 ( 6915 ) 563-573 2002.12

Research paper (scientific journal), Joint Work

　 Previous page - Next page 　