Biology subjects

Tsukamoto, T.

Publications and source records attributed to Tsukamoto, T..

14 recordsLinked to original sources

bioRxiv2025

The SPN-4 Rbfox RNA-binding protein selects maternal mRNAs for CCR4-NOT-dependent clearance in early Caenorhabditis elegans embryos

The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related SPN-4 RNA-binding protein as a specificity factor and the CCR4-NOT deadenylase complex as an effector. We biochemically identified SPN-4-associated mRNAs from late-stage oocytes and found that the set of SPN-4-associated transcripts is enriched for maternal mRNAs that undergo early decay. Single-molecule fluorescence in situ hybridization experiments established that many SPN-4-associated mRNAs fail to be eliminated in the absence of SPN-4. In the 3UTRs of two target mRNAs, we identified Rbfox motifs that are required for SPN-4-dependent clearance in vivo and bind SPN-4 in vitro. In a genetic screen to identify factors that work with SPN-4, we isolated mutant alleles of CCR4-NOT components. Auxin-induced degradation of the LET-711/NOT1 scaffold and the CCF-1 deadenylase disrupted clearance of two SPN-4-associated transcripts. Our results support a model in which SPN-4 initiates expression in late-stage oocytes, associates with maternal mRNA targets through RNA sequences in their 3UTRs and promotes CCR4-NOT-mediated decay during early embryogenesis.

developmental biology↗

bioRxiv2025

Discovery of Light-Powered Organic Ion Transport by a Natural Protein

Membrane transport proteins play vital roles in living cells by selectively transporting ions and molecules across biological membranes. Among these, microbial rhodopsins are unique in their unparalleled capacity to harness light to drive ion translocation. This distinctive feature has led to their widespread use as optogenetic tools in neuroscience, physiology, and biomed-ical applications. While not all microbial rhodopsins function as ion transporters, many ion-translocating variants have been discov-ered since the identification of the first member--a light-driven H+ pump--in the 1970s. These proteins share a compact structure composed of only seven transmembrane helices and have long been thought to specialize exclusively in transporting small inorganic ions such as H+, Cl-, and Na+. Here, we show that several anion-pumping microbial rhodopsins can also transport organic anions. In particular, a rhodopsin from cyanobacteria is capable of transporting bulky organic anions, including those containing benzene rings, with molecular volumes up to [~]120 [A]3--five times that of Cl-. These organic ions bind to the dark state and are translocated upon photoactivation, following a mechanism similar to that of inorganic anion transport. Mutational analysis indicates that both classes of substrates share a common binding site. Only anions with pKa values below 2 were transported, suggesting that a retained negative charge is essential for binding to the dark state--a prerequisite for transport. This study expands the known sub-strate repertoire of microbial rhodopsins and introduces new possibilities for optogenetic strategies based on light-driven delivery of bioactive organic molecules.

biophysics↗

bioRxiv2025

A sperm-oocyte protein partnership required for egg activation in Caenorhabditis elegans

ABSTRACTFertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. C. elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm protein, SPE-11, forms a complex with an oocyte protein, OOPS-1 (Oocyte Partner of SPE-11), and that the protein complex is required for the completion of meiosis, the block to polyspermy, and eggshell formation. Consistent with the molecular interaction of their encoded proteins, oops-1 and spe-11 exhibit identical null phenotypes, displaying defects in meiotic progression and cytokinesis. We show that the complex binds F-actin in the absence of other proteins and inhibits the nucleation of actin filaments in vitro. Thus, the OOPS-1-SPE-11 complex may function to promote F-actin-mediated meiotic cytokinesis. Both OOPS-1 and SPE-11 are intrinsically disordered proteins that are highly phosphorylated. Biochemical and genetic experiments define interactions with the protein phosphatase 1 homologs GSP-3/4, which appear to promote OOPS-1-SPE-11 function. Genetic results support a model in which the OOPS-1-SPE-11 complex interacts with the cortical EGG complex to promote meiotic cytokinesis and to activate synthesis of the eggshell.

developmental biology↗

bioRxiv2024

A Novel Support Vector Machine-Based One-Day, Single-Dose Prediction Model of Genotoxic Hepatocarcinogenicity in Rats

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment, and it also contributes to cancer prevention. This study aims to develop a one-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. This classifier effectively distinguishes GHCs from other compounds, demonstrating 100% sensitivity and over 96% specificity. To further assess the models effectiveness and reliability, we conducted multi-institutional one-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR (qPCR) 24 hours after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present one-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

pharmacology and toxicology↗

bioRxiv2022

Validating the representation of distance between infarctdiseases using Word2Vec word embedding

ObjectiveTo determine if inter-disease distances between word embedding vectors using the picot-and-cluster strategy (PCS) are a valid quantitative representation of similar disease groups in a limited domain. Materials and MethodsAbstracts were extracted from the Ichushi-Web database and subjected to morphological analysis and training using the Word2Vec. From this, word embedding vectors were obtained. For words including "infarction", we calculated the cophenetic correlation coefficient (CCC) as an internal validity measure and the adjusted rand index (ARI), normalized mutual information (NMI), and adjusted mutual information (AMI) with ICD-10 codes as the external validity measures. This was performed for each combination of metric and hierarchical clustering method. ResultsSeventy-one words included "infarction", of which 38 diseases matched the ICD-10 standard with the appearance of 21 unique ICD-10 codes. The CCC was most significant at 0.8690 (metric and method: euclidean and centroid), while the AMI was maximal at 0.4109 (metric and method: cosine and correlation, and average and weighted). The NMI and ARI were maximal at 0.8463 and 0.3593, respectively (metric and method: cosine and complete). DiscussionThe metric and method that maximized the internal validity measure were different from those that maximized the external validity measures; both produced different results. The Cosine distance should be used when considering ICD-10, and the Euclidean distance when considering the frequency of word occurrence. ConclusionThe distributed representation, when trained by Word2Vec on the "infarction" domain from a Japanese academic corpus, provides an objective inter-disease distance used in PCS.

bioinformatics↗

bioRxiv2021

Ubiquitin ligases and a processive proteasome facilitate protein clearance during the oocyte-to-embryo transition in Caenorhabditis elegans

The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hu15, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation. Article SummaryThe ubiquitin-mediated degradation of translational regulatory RNA-binding proteins is a conserved feature of the oocyte-to-embryo transition (OET). C. elegans LIN-41 is a master regulator of oogenesis and is found in a large translational regulatory ribonucleoprotein (RNP) complex with more than 1000 maternal transcripts and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11. We show that the concerted action of ubiquitin-mediated protein degradation and proteasome processivity rapidly disassemble LIN-41-containing RNPs during the OET thereby relieving repression of many maternal transcripts.

developmental biology↗

bioRxiv2021

High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

biochemistry↗

bioRxiv2020

Decreased torsinA or LINC Complex Function Rescues a Laminopathy in Caenorhabditis elegans

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex spanning the nuclear envelope. In turn, the AAA+ ATPase torsinA regulates force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog, lmn-1. Loss of OOC-5 activity prevents nuclear collapse in lmn-1 mutants by disrupting the function of the LINC complex. These results suggest that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. One Sentence SummaryInhibiting LINC complex activity prevents a progeric syndrome in C. elegans.

developmental biology↗

bioRxiv2019

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3 splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. Author SummaryMutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal that multiple sacy-1/DDX41 missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicing-independent and splicing-dependent alterations in the transcriptome.

genetics↗

bioRxiv2018

Age of onset in genetic prion disease and the design of preventive clinical trials

Regulatory agencies worldwide have adopted programs to facilitate drug development for diseases where the traditional approach of a randomized trial with a clinical endpoint is expected to be prohibitively lengthy or difficult. Here we provide quantitative evidence that this criterion is met for the prevention of genetic prion disease. We assemble age of onset or death data from N=1,094 individuals with high penetrance mutations in the prion protein gene (PRNP), generate survival and hazard curves, and estimate statistical power for clinical trials. We show that, due to dramatic and unexplained variability in age of onset, randomized preventive trials would require hundreds or thousands of at-risk individuals in order to be statistically powered for an endpoint of clinical onset, posing prohibitive cost and delay and likely exceeding the number of individuals available for such trials. Instead, the characterization of biomarkers suitable to serve as surrogate endpoints will be essential for the prevention of genetic prion disease. Biomarker-based trials may require post-marketing studies to confirm clinical benefit. Parameters such as longer trial duration, increased enrollment, and the use of historical controls in a post-marketing study could provide opportunities for subsequent determination of clinical benefit.

neuroscience↗

bioRxiv2018

Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein to Ensure A Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1. Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF-type ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two non-overlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sites is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M-phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte mRNA targets. Based on these observations, we propose a molecular model for the elimination of LIN-41 by SCFSEL-10 and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

developmental biology↗

bioRxiv2017

Loss of bone marrow cells including CD34+ cells following CCR5-tropic HIV-1 infection of humanized NOD/SCID/JAK3null mice and partial protection of those cells by a promotor-targeting shRNA

ObjectivesHematological abnormalities that include changes in bone marrow, such as in anemia and pancytopenia, are common among human immunodeficiency virus (HIV)-infected patients, particularly in the advanced stage of disease. This study aimed to provide better experimental evidence of such manifestations in animal models.\n\nDesignNOD/SCID/JAK3null (NOJ) mice were transplanted with human cord-derived CD34+ cells with or without transduction with a lentiviral vector expressing a promoter-targeting shRNA called PromA.\n\nMethodsAt 16 weeks after transplantation, mice engrafted with CD34+ cells were infected with CCR5-tropic HIV-1JRFL.\n\nResultsAt week 2 post infection, HIV replication was observed in peripheral blood mononuclear cells and splenocytes. In mice transplanted with unmanipulated CD34+ cells, viral replication was accompanied by a loss of peripheral/spleen CD4+CCR5+ T cells. Interestingly, bone marrow CD34+ cells in HIV-infected mice were also depleted, but in a CXCR4-associated manner. Conversely, the lentiviral transfer of PromA in CD34+ cells prior to transplantation rendered the humanized NOJ mice resistant to HIV replication in CD4+ T cells, resulting in better preservation of peripheral/spleen CD4+CCR5+ T cells and bone marrow CD34+ cells at two weeks after infection.\n\nConclusionsThese results implicate the importance of evaluating hematopoietic stem/progenitor cell pools in addition to peripheral CD4+ T-cell counts to assess the early stage of HIV infection. Moreover, stable gene transfer of PromA to hematopoietic stem cells not only limited HIV replication but also led to preservation of different subsets of hematopoietic cells, including bone marrow stem/progenitor cells.

microbiology↗

bioRxiv2017

HIV Persistence Alters CXCR4 Expression Patterns In OP9-DL1-Mediated Differentiation Of In Vitro Infected Human Cord Blood CD34+ Cells

HIV-1 causes the loss of CD4+ T cells via depletion or impairment of their production. The latter involves infection of thymocytes, but the involvement of other cells including haematopoietic CD34+ cells remains unclear even though HIVpositive patients frequently manifest myelosuppression. In this study, we utilised the OP9-DL1 coculture system, which supports in vitro T-lineage differentiation of human haematopoietic stem/progenitor cells. Cord-derived CD34+ cells were infected with CXCR4-tropic HIV-1NL4-3 and cocultured. HIV-infected cocultures exhibited sustained viral replication for 5 weeks, as well as reduced CD4+ T-cell growth at weeks 3-5. We further revealed that CD34+CD7+CXCR4+ cells can be quickly depleted as early as in 1 week after infection of the subset, and this was accompanied by the emergence of CD34+CD7+CD4+ cells. These results indicate that CXCR4-tropic HIV-1 strains may disrupt CD34+CD7+ lymphoid progenitor cell pools, presumably leading to impaired T-cell production potential.

microbiology↗

bioRxiv2017

Use Of RetroNectin In Studies Requiring In Vitro HIV Infection Of Human Hematopoietic Stem/Progenitor Cells

Human immunodeficiency virus (HIV) causes damage, directly or indirectly, to the whole hematopoietic system including CD34+ hematopoietic stem/progenitor cells (HSPC). CXCR4-tropic strains of HIV-1 may be potent to affect the function of CD34+CXCR4+ progenitor cells either by infecting the cells or by modifying the dynamics of more differentiated hematopoietic cells. However, CD34+ cells are known for the resistance to HIV-1 infection in vitro, restricting the detailed analysis of the impact of HIV upon HSPC. Here the authors report a use of RetroNectin, a recombinant fibronectin fragment used for gene transfer with lentiviral vectors, to overcome the limitation.

microbiology↗