Biology subjects

Nakagawa, S.

Publications and source records attributed to Nakagawa, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Macrophage innate immune gene expression requires dynamic regulation of the nuclear paraspeckle

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages needs to be exquisitely well-controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h post-stimulation) and disaggregate (2h post-stimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. Expression of a large cohort of cytokines, chemokines, and antimicrobial mediators is compromised in lipopolysaccharide-treated macrophages lacking Neat1, resulting in a failure to express a cohort of pro-inflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reprograms the nuclear RNA binding protein landscape to enable inflammatory gene expression following innate stimuli. SIGNIFICANCE STATEMENTTo mount appropriate immune responses and fight infection, macrophages need to sense and respond to pathogen-associated signals with incredible precision. Membraneless organelles (MLOs) are complexes of RNAs and proteins that change in size, shape, and abundance in response to extracellular signals. We hypothesized that an MLO called the nuclear paraspeckle helps macrophages initiate and calibrate innate immune gene expression during infection. We found that paraspeckles rapidly aggregate and then dissolve in macrophages following pathogen sensing. Macrophages lacking paraspeckles cannot properly induce inflammatory genes, resulting in a failure to control replication of intracellular bacterial and viral pathogens. These data suggest that altered paraspeckle dynamics may dysregulate inflammatory gene expression in a variety of human diseases.

immunology↗

bioRxiv2023

Distinct mechanisms for sebaceous gland self-renewal and regeneration provide durability in response to injury

Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single cell RNA-sequencing, we uncovered both direct and indirect paths by which these resident SG progenitors ordinarily differentiate into sebocytes, including transit through a PPAR{gamma}+Krt5+ transitional cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.

developmental biology↗

bioRxiv2023

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

developmental biology↗

bioRxiv2023

No general effects of advancing male age on ejaculates: a meta-analysis across the animal kingdom

Senescence, the deterioration of organismal function with advancing age, is a puzzling biological phenomenon. While actuarial senescence (i.e., age-dependent increases in mortality rates) is well described across some taxa, reproductive senescence (i.e. age- dependent declines in reproduction) is less understood, especially in males, with mixed patterns reported across studies. To examine the evidence for male reproductive senescence, we investigated how advancing male age affects ejaculate traits across non-human animals via a meta-analysis yielding 1814 effect sizes from 379 studies. We found no evidence for a general pattern of reproductive senescence. Instead, we found high heterogeneity for how reproduction changes with male age across animals. Some of this heterogeneity (>10%) was associated with biological factors. For example, there were taxonomical differences for some ejaculate traits -- sperm motility declined with male age in lab rodents and fish, whereas ejaculate size improved with male age in bulls, fish, and insects. Some methodological factors were also important in explaining this heterogeneity: studies sampling a larger proportion of a species lifespan were more likely to detect senescence in ejaculate traits, emphasising the need to examine the full life cycle of species to document senescence. Contrary to predictions, we reveal that the evidence for senescence in ejaculate traits is sporadic. Our findings will help generate novel hypotheses and identify more effective methodological approaches for studying male reproductive senescence.

evolutionary biology↗

bioRxiv2023

Delivering mRNAs to mouse tissues using the SEND system

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo. Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

genomics↗

bioRxiv2022

Dynamic evolution of retroviral envelope genes in egg-laying mammalian genomes

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino-acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

genomics↗

bioRxiv2022

Taxonomy of interventions at academic institutions to improve research quality

Research institutions and researchers have become increasingly concerned about poor research reproducibility and replicability, and research waste more broadly. Research institutions play an important role and understanding their intervention options is important. This review aims to identify and classify possible interventions to improve research quality, reduce waste, and improve reproducibility and replicability within research-performing institutions. Taxonomy development steps: 1) use of an exemplar paper of journal-level research quality improvement interventions, 2) 2-stage search in PubMed using seed and exemplar articles, and forward and backward citation searching to identify articles evaluating or describing research quality improvement, 3) elicited draft taxonomy feedback from researchers at an open-sciences conference workshop, and 4) cycles of revisions from the research team. The search identified 11 peer-reviewed articles on relevant interventions. Overall, 93 interventions were identified from peer-review literature and researcher reporting. Interventions covered before, during, and after study conduct research stages and whole of institution. Types of intervention included: Tools, Education & Training, Incentives, Modelling & Mentoring, Review & Feedback, Expert involvement, and Policies & Procedures. Identified areas for research institutions to focus on to improve research quality and for further research includes improving incentives to implement quality research practices, evaluating current interventions, encourage no- or low-cost/high-benefit interventions, examine institution research culture, and encourage mentor-mentee relationships.

scientific communication and education↗

bioRxiv2022

SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level. HighlightsO_LIOmicron spike receptor binding domain determines virological characteristics C_LIO_LISpike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron C_LIO_LIAcquisition of the spike S375F mutation triggered the explosive spread of Omicron C_LIO_LIF375-H505-mediated {pi}-{pi} interaction in the spike determines the phenotype of Omicron C_LI

microbiology↗

bioRxiv2022

Sex differences in allometry for phenotypic traits indicate that females are not scaled males

Sex differences in the lifetime risk and expression of disease are well-known. Preclinical research targeted at improving treatment, increasing health span, and reducing the financial burden of health care, has mostly been conducted on male animals and cells. The extent to which sex differences in phenotypic traits are explained by sex differences in body weight remains unclear. We quantify sex differences in the allometric relationship between trait value and body weight for 375 phenotypic traits in male and female mice, recorded in >2.1 million measurements from the International Mouse Phenotyping Consortium. We find sex differences in allometric parameters (slope, intercept, residual SD) are common (76% traits). Body weight differences do not explain all sex differences in trait values but scaling by weight may be useful for some traits. Our results support a trait-specific patterning of sex differences in phenotypic traits, promoting case-specific approaches to drug dosage scaled by body weight.

evolutionary biology↗

bioRxiv2022

Species-Specific Formation of Paraspeckles in Intestinal Epithelium Revealed by Characterization of NEAT1 in Naked Mole-rat

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2. The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2, that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

molecular biology↗

bioRxiv2022

Monotreme-specific conserved proteins derived from retroviral reverse transcriptase

Endogenous retroviruses (ERVs) have played an essential role in the evolution of mammals. Many ERV-derived genes are reported in the therians that are involved in placental development. However, the contribution of the ERV-derived genes in monotremes, which are oviparous mammals, remains to be uncovered. Here, we conducted a comprehensive search for possible ERV-derived genes in platypus and echidna genomes and identified three reverse transcriptase-like genes named RTOM1, 2, and 3. They were found to be clustered in the GRIP2 intron. Phylogenetic analysis revealed that RTOM1, 2, and 3 are strongly conserved between these species, and they were generated by tandem duplications before the divergence of platypus and echidna. The RTOM transcripts were specifically expressed in the testis, suggesting the physiological importance of RTOM genes. This is the first study reporting monotreme-specific de novo gene candidates derived from ERVs, which provides new insights into the unique evolution of monotremes.

genomics↗

bioRxiv2022

NeoRdRp: A comprehensive dataset for identifying RNA-dependent RNA polymerase of various RNA viruses from metatranscriptomic data

RNA viruses are distributed throughout various environments, and most RNA viruses have recently been identified by metatranscriptome sequencing. However, due to the high nucleotide diversity of RNA viruses, it is still challenging to identify novel RNA viruses from metatranscriptome data. To overcome this issue, we created a dataset of RNA-dependent RNA polymerase (RdRp) domains that are essential for all RNA viruses belonging to Orthornavirae. Genes with RdRp domains from various RNA viruses were clustered based on their amino acid sequence similarity. For each cluster, a multiple sequence alignment was generated, and a hidden Markov model (HMM) profile was created if the number of sequences was greater than three. We further refined the 426 HMM profiles by detecting the RefSeq RNA virus sequences and subsequently combined the hit sequences with the RdRp domains. As a result, a total of 1,182 HMM profiles were generated from 12,502 RdRp domain sequences, and the dataset was named NeoRdRp. Almost all NeoRdRp HMM profiles successfully detected RdRp domains, specifically in the UniProt dataset. Furthermore, we compared the NeoRdRp dataset with two previously reported methods for RNA virus detection using metatranscriptome sequencing data. Our methods successfully identified most of the RNA viruses in the datasets; however, some RNA viruses were not detected, as in the cases of the other two methods. The NeoRdRp can be repeatedly improved by adding new RdRp sequences and is applicable as a system for detecting various RNA viruses from diverse metatranscriptome data.

microbiology↗

bioRxiv2021

Ineffective neutralization of the SARS-CoV-2 Mu variant by convalescent and vaccine sera

On August 30, 2021, the WHO classified the SARS-CoV-2 Mu variant (B.1.621 lineage) as a new variant of interest. The WHO defines "comparative assessment of virus characteristics and public health risks" as primary action in response to the emergence of new SARS-CoV-2 variants. Here, we demonstrate that the Mu variant is highly resistant to sera from COVID-19 convalescents and BNT162b2-vaccinated individuals. Direct comparison of different SARS-CoV-2 spike proteins revealed that Mu spike is more resistant to serum-mediated neutralization than all other currently recognized variants of interest (VOI) and concern (VOC). This includes the Beta variant (B.1.351) that has been suggested to represent the most resistant variant to convalescent and vaccinated sera to date (e.g., Collier et al, Nature, 2021; Wang et al, Nature, 2021). Since breakthrough infection by newly emerging variants is a major concern during the current COVID-19 pandemic (Bergwerk et al., NEJM, 2021), we believe that our findings are of significant public health interest. Our results will help to better assess the risk posed by the Mu variant for vaccinated, previously infected and naive populations.

microbiology↗

bioRxiv2021

SARS-CoV-2 Lambda variant exhibits higher infectivity and immune resistance

SARS-CoV-2 Lambda, a new variant of interest, is now spreading in some South American countries; however, its virological features and evolutionary trait remain unknown. Here we reveal that the spike protein of the Lambda variant is more infectious and it is attributed to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino-acid deletion mutation in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the insertion of the RSYLTPGD246-253N mutation is closely associated with the massive infection spread of the Lambda variant in South America. HighlightsO_LILambda S is highly infectious and T76I and L452Q are responsible for this property C_LIO_LILambda S is more susceptible to an infection-enhancing antibody C_LIO_LIRSYLTPGD246-253N, L452Q and F490S confer resistance to antiviral immunity C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2021

Identification of the cell-type-specific ER membrane protein Tanmp expressed in hypothalamic tanycytes and subsets of neurons

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. During a screening of novel noncoding RNAs abundantly expressed in mouse brains, we incidentally identified a gene termed Tanmp, which encoded an endoplasmic reticulum (ER) protein without known functional domains. Tanmp is specifically expressed in the nervous system, and the highest expression was observed in a specialized cell type called tanycyte that aligns the ventral wall of the third ventricle in the hypothalamus. Immunostaining of Tanmp revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that Tanmp associates with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking Tanmp did not exhibit overt phenotypes, suggesting that Tanmp is not essential in mice reared under normal laboratory conditions. We also found that RNA probes that are predicted to uniquely detect Tanmp mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

cell biology↗

bioRxiv2021

SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and currently, four variants of concerns (VOCs) are considered as the hazardous SARS-CoV-2 variants to the human society1. The newly emerging VOC, the B.1.617.2/Delta variant, closely associates with a huge COVID-19 surge in India in Spring 20212. However, its virological property remains unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic, and notably, more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates the spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than the parental virus. Our data suggest that the P681R mutation is a hallmark that characterizes the virological phenotype of the B.1.617.2/Delta variant and is closely associated with enhanced pathogenicity.

microbiology↗

bioRxiv2021

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

molecular biology↗

bioRxiv2021

Uhrf1 governs the proliferation and differentiation of muscle satellite cells

DNA methylation is an essential form of epigenetic regulation responsible for cellular identity. In muscle stem cells, termed satellite cells, DNA methylation patterns are tightly regulated during differentiation. However, it is unclear how these DNA methylation patterns are maintained. We demonstrate that a key epigenetic regulator, ubiquitin like with PHD and RING finger domains 1 (Uhrf1), is activated in proliferating myogenic cells but not expressed in quiescent or differentiated myogenic cells in mice. Ablation of Uhrf1 in mouse satellite cells impairs their proliferation and differentiation, leading to failed muscle regeneration. Loss of Uhrf1 in satellite cells alters transcriptional programs, leading to DNA hypomethylation with activation of Cdkn1a and Notch signaling. Although down-regulation of Cdkn1a rescued proliferation but not differentiation, inhibition of Notch signaling rescued impaired differentiation of Uhrf1-deficient satellite cells. These findings point to Uhrf1 as a regulator of self-renewal and differentiation of satellite cells via genome-wide DNA methylation patterning.

molecular biology↗