Biology subjects

Nakanishi, K.

Publications and source records attributed to Nakanishi, K..

8 recordsLinked to original sources

bioRxiv2026

Axolotl regeneration reveals a dormant cis-regulatory grammar conserved across vertebrate genomes

Unlike most mammals, which lack the capacity to regenerate complex tissues following injury, other vertebrates such as the axolotl rebuild complete limbs throughout life, yet the regulatory mechanisms underlying this striking difference have remained elusive. Here, we define the core cis-regulatory motif grammar driving axolotl limb regeneration and demonstrate that this grammar is conserved within the syntenic neighborhoods of regeneration-gene orthologs in human and mouse genomes, despite being epigenetically sealed in adult mammalian tissues. Integrating this cross-species grammar projection with AlphaGenome, a multimodal genomic AI capable of predicting epigenomic states from long sequence context, we find that the highest-ranking candidate loci are predicted to occupy a state of bivalent dormancy marked by the co-enrichment of poising and repressive histone modifications alongside suppression of transcriptional activity and chromatin accessibility. Systematic in silico motif perturbation further predicts that this dormant state is actively enforced by specific dormancy-stabilizing sequence elements, and that disrupting these elements shifts candidate loci toward a regeneration-competent chromatin configuration. Our findings support a model in which the regenerative blueprint has not been erased from the mammalian genome but locked within it, opening new avenues for understanding the evolution of regenerative competence and for the rational reactivation of latent regenerative programs.

systems biology↗

bioRxiv2026

Integrating virtual cell modeling with patient-derived transcriptomics to uncover target signals in rheumatoid arthritis

Rheumatoid arthritis affects millions of people worldwide, yet a substantial fraction of patients fail to achieve lasting benefit from current therapies. AI-based virtual cell models offer a promising route to simulate the effects of thousands of potential drug targets computationally, but whether their predictions genuinely reflect what happens in the disease-relevant cells of patients undergoing treatment has been unclear. Here, we introduce ImmunoSTATE, a clinically anchored framework that benchmarks empirical and virtual perturbations directly against patient-derived treatment trajectories in pathogenic CXCL13+ synovial T cells from nine patients with rheumatoid arthritis sampled before and after treatment. Empirical genome-wide CRISPR interference screening prioritizes T cell receptor (TCR)-proximal signaling over the measurable components of the JAK-STAT pathway for transcriptomic reversal of the disease signature, a result that remained robust across leave-one-patient-out reanalyses. Comparison of virtual perturbation signals across healthy donor CD4+ T cells and rheumatoid-arthritis patient cells reveals broad preservation of target priorities, together with selected disease-context-enriched shifts, including stronger prioritization of SOX4 in patient cells. ImmunoSTATE provides a clinically anchored framework for evaluating virtual cell models in disease-relevant immune contexts and demonstrates that distinguishing training-set status and cellular context are essential steps in the reliable interpretation of virtual perturbation outputs.

immunology↗

bioRxiv2025

CRISPR/Cas9-mediated genome editing reveals the involvement of a polyphenol oxidase in the shikonin-specific biosynthesis in Lithospermum erythrorhizon

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon, only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3''-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

biochemistry↗

bioRxiv2024

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgo) and the DNA Defense Module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

biophysics↗

bioRxiv2023

Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, central, 3' supplementary, and tail regions across the loaded guide, enabling the RISC to recognize and cleave target RNAs. This guide segmentation is caused by anchoring the 3' end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because there was no method by which to do so. Using a 3'[->]5' exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3' ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3 ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.

biochemistry↗

bioRxiv2022

Manganese-dependent microRNA trimming by 3'->5' exonucleases generates 14-nucleotide or shorter tiny RNAs

MicroRNAs (miRNAs) are about 22-nucleotide (nt) non-coding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3[->]5 exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3hExo), capable of trimming AGO-associated full-length miRNAs to 14 nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

molecular biology↗

bioRxiv2021

biPACT: a method for three-dimensional visualization of mouse spinal cord circuits of long segments with high resolution

BackgroundThe spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injuries. Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging. New MethodWe present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord. ResultsCorticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading, Comparison with Existing MethodsThe present method does not require special equipment, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. It enables to visualize individual neural fiber of specific neural circuit in long spinal cord segments. ConclusionsBy using simple neural staining, and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord. HighlightsO_LINo special devices or genetic tracers are required for a new clearing method C_LIO_LINeuronal fibers are individually depicted in long segments of mouse spinal cord. C_LIO_LIRe-staining of neuronal fiber is possible. C_LIO_LIStereotaxic observation is achieved by 3-D reconstruction with open-source software. C_LI

neuroscience↗

bioRxiv2020

Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

RNA interfering is a eukaryote-specific gene silencing by 20[~]23 nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3' end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer by an [~] 82-fold increase in target cleavage. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

biochemistry↗