Biology subjects

Gotoh, Y.

Publications and source records attributed to Gotoh, Y..

31 records · Page 2Linked to original sources

bioRxiv2022

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance

Glutamine synthetase is the key enzyme of nitrogen assimilation, which is encoded in the first cistron of glnALG operon and is induced under nitrogen limiting conditions through transcriptional activation by NtrBC in Salmonella and E. coli. 2-oxoglutarate serves as the carbon skeleton of glutamate and glutamine, but how 2-oxoglutarate fluctuation is controlled in response to nitrogen availability remained unknown. We show that the glnA mRNA produces an Hfq-dependent GlnZ sRNA from its 3 sUTR through RNase E-mediated cleavage. Through a base-pairing mechanism, GlnZ primarily regulates sucA, encoding the E1o component of 2-oxoglutarate dehydrogenase. In the cells grown on glutamine as the nitrogen source, the endogenous GlnZ represses the expression of SucA to redirect the carbon flow from the TCA cycle to the nitrogen assimilation pathway. This study also clarifies that the release of GlnZ sRNA from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA, and thus the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

microbiology↗

bioRxiv2021

Three-dimensional chromatin architecture of early-stage mouse embryos reconstructed via recurrence plots

The chromatin conformation capture-related methods such as Hi-C have improved our understanding of nuclear architecture and organization in recent years. However, reconstruction of nuclear architecture from single-cell Hi-C (scHi-C) data is challenging due to limited information of DNA contacts obtained from a single cell. We have previously developed the Recurrence Plot-Based Reconstruction (RPR) method for reconstructing three dimensional (3D) genomic structure from Hi-C data of single haploid cells (1) and diploid cells (2). This algorithm is based on a recurrence plot, a tool of nonlinear time-series analysis for visualizing patterns within a time series (3, 4), and enables the reconstruction of a unique 3D chromosome architecture even from low-coverage DNA contact information. Here we used the RPR method to analyzing published scHi-C data of diploid cells derived from early-stage F1 hybrid embryos (5) as a proof-of-concept for understanding of global developmental changes in chromosomal architecture of early stage embryos. We found that paternal and maternal chromosomes become gradually intermingled from 1 cell to 64 cell stage, and that discrete chromosome territories are largely established between 8 cell and 64 cell stages. We also observed Rabl-like polarization of chromosomes from the 2-to 8-cell stages, but this polarization becomes mostly dissolved by the 64-cell stage. Rabl-like chromosome polarization precedes rod-like extension and parallel alignment of chromosomes, implicating the role of Rabl-like polarization in effective mixing of chromosomes before establishing chromosome territories. We also found cell-to-cell variability in chromatin configuration. A combination of scHi-C and RPR analyses can depict features of the 3D chromatin architecture of individual cells at different developmental stages during early embryogenesis.

developmental biology↗

bioRxiv2021

Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Mimiviridae, and prokaryotes in a coastal ecosystem

Coastal seawater is the habitat of different microbial communities. These communities are affected by seasonal environmental changes and fluctuating nutrient availability, as well as competitive and cooperative interspecific interactions. In this work, we investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses infecting eukaryotes (i.e. Mimiviridae), as well as prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and the Mimiviridae polB gene as marker genes in 43 seawater samples collected during 20 months. Communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles for eukaryotes, Mimiviridae, and prokaryotes. However, the community dynamics showed intriguing differences in several aspects such as the recovery rate after a year. We further show that the differences in the community dynamics can be explained by differences in the recurrence/persistence levels of individual ASVs among eukaryotes, Mimiviridae, and prokaryotes. Mimiviridae ASVs were less persistent than eukaryotic ASVs, and prokaryotic ASVs were the most persistent. We argue that the differences in the specificity of interactions (i.e. virus-eukaryote vs prokaryote-eukaryote) as well as the survival strategies are at the origin of the distinct community dynamics among eukaryotes, Mimiviridae, and prokaryotes. One sentence summaryA one year observation of coastal microbial communities revealed similar but different community dynamics for eukaryotes, a group of large viruses, and prokaryotes.

microbiology↗

bioRxiv2020

Prophages integrating into prophages: a mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 PPs (up to 21 PPs), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present prophages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the prophages integrated into prophages are Stx-encoding prophages and have induced the duplication of Stx-encoding phages in a single cell. The identified att sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different att sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These "prophages-in-prophages" represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx-encoding prophages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species. Author summaryMultiple prophages are often integrated in a bacterial host chromosome and some are present at a single locus in tandem. The most striking examples are Shiga toxin (Stx)-producing and enteropathogenic Escherichia coli (STEC and EPEC) strains, which usually contain more than 10 prophages (up to 21). Many of them encode a cluster of type III secretion system (T3SS) effector genes, contributing the acquisition of a large number of effectors (>30) by STEC and EPEC. Here, we describe prophages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Two or three different attachment sequences derived from host chromosomes are present in some prophages, generating prophage clusters in various complex configurations. Of note, some of such prophages-in-prophages are Stx-encoding prophages and have induced the duplication of Stx-encoding prophages. Thus, these "prophages-in-prophages" represent an important inter-phage interaction as they can promote not only the accumulation of T3SS effectors in STEC and EPEC but also the duplication of Stx-encoding prophages and the conversion of EPEC to STEC.

microbiology↗

bioRxiv2020

Gene expression changes of seven stonefly species in responses to a latitudinal-environmental gradient.

Latitudinal variation has been known to create strong selection pressure for genomic variation that enables the adaptation and survival of organisms. By altering gene expression patterns, organisms can modify their adaptive potential to heterogeneous environmental conditions along a latitudinal gradient; however, there is a gap in our understanding of how physiological consequences in wild species are affected and how changing environmental conditions act on multiple species. Here, we investigated how seven stream stonefly species sampled from four geographical regions in Japan differ in their responses to latitudinal variations by measuring gene expression (RNA-sequencing) differences within species and gene co-expression among species. We found that a large number of genes (622) were differentially expressed along the latitudinal gradient. The high species-specific gene expression diversity found at higher latitude regions was probably associated with low temperatures and high water discharge, which suggests the adaptive potential of stonefly specie. In contrast, similar gene expression patterns among species was observed at lower latitudes, which suggests that strong environmental stress occurs in warmer regions. Weighted gene co-expression network analysis (WGCNA) identified 22 genes with similar expression patterns among species along the latitudinal gradient. Among the four geographical regions, high differential expression patterns in the co-expressed genes from two regions were found, suggesting that the local environment strongly affects gene expression patterns among species in these regions. Respiration, metabolism, and developmental co-expressed genes exhibited a latitudinal cline, showing clear evidence of divergent adaptive responses to latitude. Our findings demonstrate that stonefly species are differentially adapted to local environmental conditions, and imply that adaptation in gene expression could be shared by multiple species under environmental stress conditions. This study highlights the importance of considering multiple species when evaluating the consequences of environmental changes on aquatic insect communities, and possible mechanisms to cope with environmental changes.

molecular biology↗

bioRxiv2020

In utero gene transfer system for embryos before neural tube closure reveals a role for Hmga2 in the onset of neurogenesis

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is the fundamental obstacle in understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, a period for the drastic development of many tissues. In this study, we present a simple technique for transferring vectors into the endo- and ectodermal cells of mouse embryos at E7.0 or E8.0 via in utero injection, without any specialized equipment. Using this technique, introduction of retroviruses can facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. As such, this technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.

developmental biology↗

bioRxiv2020

NUDT21 links mitochondrial IPS-1 to RLR-containing stress granules and activates host antiviral defense

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid- inducible protein-I (RIG-I)-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms including type I interferon (IFN) induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. Here we show that NUDT21, an RNA binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with poly(I:C), a mimic of viral double-stranded RNA. We found that, despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.

immunology↗

bioRxiv2020

A functional RNA structure in the influenza A virus ribonucleoprotein complex for segment bundling

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.

microbiology↗

bioRxiv2020

A universal subcuticular bacterial symbiont of a coral predator, the crown-of-thorns starfish

BackgroundPopulation outbreaks of the crown-of-thorns starfish (Acanthaster planci sensu lato; COTS), a primary predator of reef-building corals in the Indo-Pacific Ocean, are major concerns in coral reef management. While biological and ecological knowledge of COTS has been accumulating since the 1960s, little is known about its associated bacteria. The aim of this study was to provide fundamental information on dominant COTS-associated bacteria through a multifaceted molecular approach. MethodsA total of 205 COTS individuals from 17 locations throughout the Indo-Pacific Ocean were examined for the presence of COTS-associated bacteria. We conducted 16S rRNA metabarcoding of COTS to determine the bacterial profiles of different parts of the body, and generated a full-length 16S rRNA gene sequence from a single dominant bacterium, which we designated COTS27. We performed phylogenetic analysis to determine the taxonomy, screening of COTS27 across the Indo-Pacific, FISH to visualize it within the COTS tissues, and reconstruction of the chromosome from the hologenome sequence data. ResultsWe discovered that a single bacterium exists at high densities in the subcuticular space in COTS forming a biofilm-like structure between the cuticle and the epidermis. COTS27 belongs to a clade that presumably represents a distinct order (so-called marine spirochetes) in the phylum Spirochaetes and is universally present in COTS throughout the Indo-Pacific Ocean. The reconstructed genome of COTS27 includes some genetic traits that are probably linked to adaptation to marine environments and evolution as an extracellular endosymbiont in subcuticular spaces. ConclusionsCOTS27 can be found in three allopatrically speciated COTS species, ranging from northern Red Sea to the Pacific, implying that symbiotic relationship arose before the speciation (approximately 2 million years ago). The universal association of COTS27 with COTS and nearly mono-specific association at least with the Indo-Pacific COTS potentially provides a useful model system for studying symbiont-host interactions in marine invertebrates.

microbiology↗

bioRxiv2019

Role of the imprinted allele of the p57Kip2 gene in mouse neocortical development

Imprinted genes are expressed from only one allele in a parent of origin-specific manner. The cyclin-dependent kinase inhibitor p57Kip2 (p57) is encoded by an imprinted gene, with the paternal allele being silenced. The possible expression and function of the paternal allele of p57 have remained little studied, however. We now show that the paternal allele of the p57 gene is expressed at a low level in the developing mouse neocortex. Surprisingly, the central nervous system-specific conditional deletion of the paternal allele (pat cKO) at the p57 locus resulted in a marked reduction in brain size. Furthermore, pat cKO gradually reduced the number of neural stem-progenitor cells (NPCs) during neocortical development, and thus reduced the number of upper-layer neurons, which were derived from late-stage NPCs. Our results thus show that the paternal allele of the p57 locus plays a key role in maintenance of NPCs during neocortical development.

developmental biology↗

bioRxiv2019

The Polycomb group protein Ring1 regulates dorsoventral patterning of the mouse telencephalon

Patterning of the dorsal-ventral (D-V) axis of the mammalian telencephalon is fundamental to the formation of distinct functional regions including the neocortex and ganglionic eminences. Morphogenetic signaling by bone morphogenetic protein (BMP), Wnt, Sonic hedgehog (Shh), and fibroblast growth factor (FGF) pathways determines regional identity along this axis. It has remained unclear, however, how region-specific expression patterns of these morphogens along the D-V axis are established, especially at the level of epigenetic (chromatin) regulation. Here we show that epigenetic regulation by Ring1, an essential Polycomb group (PcG) protein, plays a key role in formation of ventral identity in the mouse telencephalon. Deletion of the Ring1b or both Ring1a and Ring1b genes in neuroepithelial cells of the mouse embryo attenuated expression of the gene for Shh, a key morphogen for induction of ventral identity, and induced misexpression of dorsal marker genes including those for BMP and Wnt ligands in the ventral telencephalon. PcG protein-mediated trimethylation of histone H3 on lysine-27 (H3K27me3) was also apparent at BMP and Wnt ligand genes in wild-type embryos. Importantly, forced activation of Wnt or BMP signaling repressed the expression of Shh in organotypic and dissociated cultures of the early-stage telencephalon. Our results thus indicate that epigenetic regulation by PcG proteins--and, in particular, that by Ring1-- confers a permissive state for the induction of Shh expression through suppression of BMP and Wnt signaling pathways, which in turn allows the development of ventral identity in the telencephalon.

developmental biology↗

bioRxiv2018

Peroxisomes control mitochondrial dynamics and the mitochondrion-dependent pathway of apoptosis

Summary StatementsWe unveil a previously unrecognized role of peroxisomes in the regulation of mitochondrial fission-fusion dynamics, mitochondrion-dependent caspase activation, and cellular apoptosis.\n\nAbstractPeroxisomes cooperate with mitochondria in the performance of cellular metabolic functions such as fatty acid oxidation and maintenance of redox homeostasis. Whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis has remained unclear, however. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, resulted in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on dynamin-related protein 1 (Drp1). Conversely, treatment with 4-phenylbutyric acid, an inducer of peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-deficient MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, indicative of an enhanced cellular sensitivity to death signals. Together, our data unveil a previously unrecognized role of peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Given that mutations of peroxin genes are responsible for lethal disorders such as Zellweger syndrome, effects of such mutations on mitochondrion-dependent apoptosis may contribute to disease pathogenesis.

cell biology↗

bioRxiv2018

A Chemogenetic Platform for Spatio-temporal Control of β-arrestin Translocation and Signaling at G protein-Coupled Receptors

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC=\"FIGDIR/small/251769_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@1e4bf96org.highwire.dtl.DTLVardef@de4937org.highwire.dtl.DTLVardef@198dd33org.highwire.dtl.DTLVardef@e26f46_HPS_FORMAT_FIGEXP M_FIG C_FIG Although ligand-activated GPCRs induce both G-protein and {beta}-arrestin dependent signaling, gaining precise spatio-temporal control of {beta}-arrestin signaling has proven elusive. Here we describe a platform for specifically activating {beta}-arrestin-dependent signaling in situ. The platform, which we have dubbed \"GA-PAIR\" (GPCR/{beta}-Arrestin -Plant protein and Abscisic acid Induced Recruitment), can be controlled by the inert phytochemical S-(+)-abscisic acid (ABA). ABA induces interaction between ABI1 (ABA Insensitive 1) and PYL1 (Pyrabactin Resistance (PYR) 1-Like), two plant proteins with no mammalian counterparts. We fused ABI to the engineered human muscarinic M3 G protein-coupled receptor (hM3Dq) and PYL1 to {beta}-arrestin2. Addition of ABA induced rapid and nearly complete translocation of the PYL-{beta}-arrestin fusion protein and, importantly, induced both ERK and Akt signaling. Photo-uncaging a new photo-caged ABA analogue allowed us to gain relatively precise spatio-temporal control over {beta}-arrestin translocation. Because GA-PAIR facilitates the exclusive activation of endogenous {beta}-arrestin signaling pathways in the absence of a GPCR ligand or G protein, the GA-PAIR system will facilitate deconvoluting GPCR signaling in situ.

synthetic biology↗