Biology subjects

Kimura, S.

Publications and source records attributed to Kimura, S..

46 records · Page 3Linked to original sources

bioRxiv2021

Deceleration of cell cycle underpins a switch from proliferative- to terminal division in plant stomatal lineage

Differentiation of specialized cell types from self-renewing progenitors requires precise cell cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor meristemoid followed by the single symmetric division that creates an adjustable pore surrounded by paired guard cells. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and triggers differentiation. However, the role of cell cycle machinery in this transition remains unknown. Through time-lapse imaging, we discover that the symmetric division is slower than the asymmetric division. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a molecular brake that decelerates cell cycle during this transition. SMR4 is directly induced by MUTE and transiently accumulates in differentiating meristemoids. SMR4 physically and functionally associates with CYCD3;1 and extends G1-phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for the stomatal fate specification.

plant biology↗

bioRxiv2021

Transient ribosome slowdown at the decoding step triggers mRNA degradation independent of Znf598 in zebrafish

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame (ORF) determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role of the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analysis is required to define the codon effects on mRNA stability apart from the related cotranslational mRNA decay pathways such as those triggered by aberrant ribosome stalls. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of Znf598 and stall-dependent mRNA decay. We propose that codon-mediated mRNA decay is triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

molecular biology↗

bioRxiv2020

Oxicam-type NSAIDs enhance Agrobacterium-mediated transformation in plants

Agrobacterium-mediated transformation represents a key innovation for plant breeding and is routinely used in research and applied biology. However, for several species, the efficacy of transformation is limited. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs (NSAIDs), including tenoxicam (TNX), enhance the efficiency of Agrobacterium-mediated transient transformation in the model species Arabidopsis thaliana via leaf infiltration and can be successfully applied in analyses of the subcellular localisation of fluorescent fusion proteins. TNX acts as an inhibitor of plant immune responses and lacks similar transient transformation efficiency in a dde2/ein2/pad4/sid2 quadruple mutant background, thereby indicating that TNX increases the efficiency of Agrobacterium infection via a transient shutdown of the immune system mediated by jasmonic acid, ethylene, and salicylic acid signalling. In addition, we found that TNX enhances the efficiency of stable transformation in crops of agricultural and economic importance, such as Jatropha and maize, indicating that TNX can enhance the integration of exogenous DNA into the plant genome via an increased introduction of DNA into plant cells. Given that treatment with oxicam compounds is simple, cost effective, and has broad utility, we anticipate that this discovery will contribute to accelerating genome-editing technologies in plants.

plant biology↗

bioRxiv2020

Lung lineage transcription factor NKX2-1 epigenetically resolves opposing cell fates in vivo

Differential use of identical DNA sequences leads to distinct tissue lineages and then multiple cell types within a lineage, an epigenetic process central to progenitor and stem cell biology. The associated genomic changes, especially in native tissues, remain insufficiently understood, and are hereby addressed in the mouse lung, where the same lineage transcription factor NKX2-1 promotes the diametrically opposed alveolar type 1 (AT1) and AT2 cell fates. We show that the cell-type-specific function of NKX2-1 is attributed to its differential chromatin binding that is acquired or retained during development in coordination with partner transcriptional factors. Loss of YAP/TAZ redirects NKX2-1 from its AT1-specific to AT2-specific binding sites, leading to transcriptionally exaggerated AT2 cells when deleted in progenitors or AT1-to-AT2 conversion when deleted after fate commitment. Nkx2-1 mutant AT1 and AT2 cells gain distinct accessible sites including those of the opposite fate while adopting the gastrointestinal fate, suggesting an epigenetic plasticity larger than a transcriptional one. Our genomic analysis of single or purified cells, coupled with precision genetics, provides an epigenetic roadmap of alveolar cell fate and potential, and introduces an experimental benchmark for unraveling the in vivo function of lineage transcription factors.

developmental biology↗

bioRxiv2019

Surveying the landscape of tRNA modifications by combining tRNA sequencing and RNA mass spectrometry

Chemical modification of the nucleosides that comprise tRNAs are diverse1-3. Such modifications impact tRNA structure, stability, and mRNA decoding3,4. Although tRNA modifications are present in all kingdoms of life1, the structure, location, and extent of modifications have been systematically charted in very few organisms, in part because mapping modifications to tRNA sequences has been technically challenging. Here, we describe a new approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput tRNA-sequencing (tRNA-seq) data is coupled with chemical analysis and identification of tRNA modifications through RNA mass spectrometry (tRNA-SMS). As proof of concept, we applied this method to study tRNA modification profiles in two phylogenetically close bacteria, E. coli and Vibrio cholerae. Comparative tRNA-seq enabled prediction of several V. cholerae modifications that are absent from E. coli and showed the effects of various environmental conditions on V. cholerae tRNA modification profiles. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while another results from C-to-U RNA editing, a process not described before in bacteria. By combining comparative genomics with mass spectrometry, we identified a putative N- acetyltransferase required for acacp3U acetylation. These findings demonstrate the utility of the tRNA-SMS approach for rapid characterization of tRNA modification profiles and environmental control of tRNA modification. Moreover, our identification of a new modified nucleoside and RNA editing process suggests that there are many tRNA modifications awaiting discovery.

biochemistry↗

bioRxiv2019

Jagged-1 regulates Foxp3 expression and cytokine production in CD4+ T cells

Notch ligands are present during the interactions between T cells and dendritic cells (DC) and induce a myriad of effects that facilitate the activation of T cells, including the induction of T cell regulation, survival, and cytokine production. Although the ligands Delta-like 4 and Delta-like 1 are expressed as a function of DC activation, the notch ligand Jagged-1 is constitutively expressed on DC. We sought to determine the role of Jagged-1 in the interactions between CD4+ T cells and DC. We observed that Jagged-1 regulates Foxp3 expression, and Cd11cCre+Jaggedff mice have an altered expression of Foxp3 in effector cells that arise as a result of infection with the mycobacterium Bacille Calmette-Guerin. The observed changes in Foxp3 expression were correlated with an increase in cytokine production from cultures of antigen-stimulated draining lymph nodes.

immunology↗

bioRxiv2019

The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition

Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. Here, we have performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. We found that the Lid/KDM5 histone demethylase and its partner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favor the accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. We propose that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.

genetics↗

bioRxiv2019

CRK2-mediated control of ROS production by phosphorylation of the RBOHD C-terminus in Arabidopsis

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. Functional CRK2 is required for the full elicitor-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment and substitution of S703 with alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating MAMP-triggered ROS production. One-sentence summaryCRK2 associates with and activates RBOHD to trigger MAMP-induced ROS production and reveals a novel regulatory mechanism for plant NADPH oxidases through phosphorylation of the C-terminus.

plant biology↗

bioRxiv2018

CRK2 enhances salt tolerance in Arabidopsis thaliana by regulating endocytosis and callose deposition in connection with PLDα1

High salinity has become an increasingly prevalent source of stress to which plants need to adapt. The receptor-like protein kinases (RLKs), including the cysteine-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants and are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses, however their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis thaliana. We identified CRK2 as a negative regulator of endocytosis, under both normal growth conditions and salt stress. We also established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a novel role for callose deposition, in response to increased salinity, and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins we observed specific changes in CRK2s subcellular localization in response to various stress treatments. Many of CRK2s cellular functions were dependent on phospholipase D (PLD) activity, as were the subcellular localization changes. Thus we propose that CRK2 acts downstream of PLD during salt stress to regulate endocytosis and promote callose deposition, and that CRK2 adopts specific stress-dependent subcellular localization patterns in order to carry out its functions.\n\nOne sentence summaryThe receptor-like kinase CRK2 acts in connection with PLD1 to regulate endocytosis and callose deposition at plasmodesmata, enhancing salt tolerance in Arabidopsis thaliana.

plant biology↗

bioRxiv2018

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.01% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 {+/-} 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0007% (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

cancer biology↗