Published papers

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.

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.

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.

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.

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.

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.

© 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.

© 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.

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.

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.