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Papers - Kanai, Akio

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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.

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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.

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