Biology subjects

Suzuki, Y.

Publications and source records attributed to Suzuki, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Spatial transcriptomic landscape of the Ciona adult brain: functional zonalisation and cellular composition in a sessile chordate brain and a novel insight into neural gland function

The ascidian Ciona provides a key model for understanding the evolutionary origin of the vertebrate brain. While the larval nervous system has been extensively characterized, the molecular and cellular organization of the adult neural complex remains poorly defined. Here, we generated spatial transcriptomic maps of the adult Ciona neural complex from three individuals, with four serial sections per donor, using the 10x Visium platform. Clustering-based analysis identified five major tissue domains, including the cerebral ganglion, neural gland, ciliated funnel, neural gland duct/dorsal strand, and body wall muscle. To further refine spatial resolution, we computationally reconstructed approximately 980 super-resolution gene expression maps by integrating transcriptomic measurements with histological image features. The super-resolution maps enabled precise delineation of molecular territories within the neural complex. In the cerebral ganglion, high-resolution reconstruction revealed clear molecular zonation, distinguishing the cortex and medulla. Within the cortex, the central region facing the neural gland and anteroposterior distal regions showed distinct molecular properties. In the neural gland, we identified coordinated enrichment of cell-cell interaction- and extracellular matrix-related genes, suggesting specialized structural and physiological properties. We propose that the neural gland play a pivotal role for the cerebral ganglion in maintaining homeostasis, supporting development, and providing a signaling interface, which is reminiscent of a primitive form of the choroid plexus and meninges found in vertebrates. Together, this study provides the first spatially resolved transcriptomic atlas of the adult Ciona neural complex and establishes a molecular framework for investigating functional regionalization and brain evolution in chordates.

genomics↗

bioRxiv2026

Deep Microbial Colonization in 2-Billion-Year-Old Ultramafic Rock from the Bushveld Complex

Archean cratons may provide stable microbial habitats in the deep subsurface, as evidenced by the discovery of billion-year-old crustal fluids1,2. However, the long-term habitability of these cratonic environments is uncertain, as polymetamorphic evolution in most cratons typically destroys microbial habitats through mineral reactions and porosity loss3,4. Preservation of deep microbial habitats is more likely where mantle-derived magma intruded the craton after metamorphic overprinting4. Here we report the discovery of dense microbial colonization at 814 m depth within the 2.05-billion-year-old, unmetamorphosed Bushveld Igneous Complex intrusion, South Africa5. Using advanced contamination-control protocols6,7 and synchrotron-based X-ray spectroscopy, we identified indigenous microbial cells localized at the rims of phlogopite, a hydrous phyllosilicate mineral. Our study reveals that microbial colonization is associated with Fe(III) derived from the structure of phlogopite, where the dehydrogenation likely oxidizes Fe(II) to Fe(III) coupled to H2 generation8. Despite the absence of fracture-driven fluid ingress in the unfractured rock matrix, aqueous alteration evidenced at the rims by potassium removal indicates a self-sustaining habitat driven by an internal redox gradient9. These findings demonstrate that aqueous alteration of ultramafic rocks can sustain isolated microbial life over geological timescales, significantly expanding the potential for long-term habitability on both Earth and Mars4,10.

microbiology↗

bioRxiv2026

Experimental SARS-CoV-2 infection using horseshoe bats

Horseshoe bats are known as the natural reservoir of sarbecoviruses. To understand how horseshoe bats coexist with sarbecoviruses in nature, experimental infection can provide direct evidence. However, in vivo infection studies using horseshoe bats have been lacking because of the difficulty of maintaining insectivorous bats in a laboratory setting. Here, we established a stable husbandry system for greater horseshoe bats (Rhinolophus ferrumequinum) and performed experimental infection with SARS-CoV-2. In contrast to Syrian hamsters which showed substantial viral replication, infected horseshoe bats exhibited low-level but persistent viral replication in the lung without overt disease. Histological analyses revealed that inflammatory lesions in the bat lungs were spatially restricted and temporally delayed compared with those in hamsters. Transcriptomic analyses further showed preferential activation of tissue repair pathways but limited inflammatory responses following infection. Notably, bats expressed several interferon-stimulated genes prior to infection. Our results suggest that a host strategy combining constitutive antiviral state, limited inflammation and enhanced tissue repair may result in controlled viral replication without overt disease, likely enabling horseshoe bats to coexist with sarbecoviruses.

microbiology↗

bioRxiv2026

Digital twins of upright stance reveal mechanistic bifurcations underlying Parkinsonian sway phenotypes

How the central nervous system maintains upright stance--and why this capability collapses in Parkinsons disease (PD)--remains a fundamental open question in computational neuroscience. A primary barrier is a severe topological degeneracy between observable sway kinematics (z-space) and latent neural control policies (-space), causing radically distinct control strategies to mask as identical sway patterns. Here, we establish a dynamical digital twin framework bridging empirical sway time series with mechanistic physical models. Assimilating a large cohort (N = 1, 038) into an intermittent control model via Bayesian inference, we construct a bidirectional mapping (z {leftrightarrow} ) that unmasks these hidden dynamics. We prove that healthy stance is universally governed by flexible intermittent control near an optimal intermittency ratio ({rho} {approx} 0.5), whereas PD progression reflects a structural regression toward rigid continuous control ({rho} [->] 1). Crucially, parameter topology in -space reveals that neural control policies reside on a folded low-dimensional manifold separated by a distinct parameter gap; attractor bifurcation analysis demonstrates that a catastrophic tipping point is embedded within this void, proving that parkinsonian postural breakdown is driven by a discontinuous dynamical phase transition rather than continuous control decay. By unmasking hidden disease severity beneath degenerate sway phenotypes, our framework reframes parkinsonian motor failure as an attractor bifurcation on a neural control manifold, providing a modern computational realization of the classical "dynamical disease" paradigm. SignificancePostural collapse in Parkinsons disease presents a profound neurobiological paradox: cellular degeneration progresses continuously over decades, yet clinical motor failure strikes as a sudden, catastrophic collapse. We resolve this paradox by demonstrating that motor breakdown is not a passive decay of control resources, but a discontinuous dynamical phase transition (attractor bifurcation) triggered when underlying neural control policies cross a latent parameter gap. By unmasking "phenotypic degeneracy"--where radically different neural control strategies produce identical body sway--our digital twin framework redefines neurodegenerative motor failure as a qualitative regime shift in complex control dynamics, offering a universal paradigm for predicting catastrophic transitions in neurological disorders.

neuroscience↗

bioRxiv2025

Cellular responses to FGF1 are modulated by palmitoylation of the docking protein FRS2α

An important mechanism by which receptor tyrosine kinases (RTKs) mediate cellular responses involves the formation of signaling complexes through direct interactions with membrane-associated docking proteins, followed by phosphorylation of multiple tyrosine residues. These docking proteins recruit and activate downstream signaling molecules and enzymes following ligand stimulation. The docking protein FRS2 has been established as a major signaling hub activated by fibroblast growth factors (FGFs), neurotrophic factors, and other extracellular cues. Here, we show that palmitoylation of FRS2 at two sites is essential for stabilizing its myristoylation-dependent association with the plasma membrane. FGF1-induced MAPK activation and other cellular responses are partially restored in cells expressing FRS2 mutants deficient in either one of the two palmitoylation sites. However full restoration of signal strength including MAPK response and other FGF1-induced cellular activities requires palmitoylation at both FRS2 sites. In addition to enhancing signaling robustness, anchoring of FRS2 to the plasma membrane creates a structural platform for assembling multi-protein complexes essential for cytoskeletal reorganization associated with membrane ruffling, macropinocytosis, and other FGF1-induced processes. Finally, we demonstrate that while PC12 cells lacking FRS2 or deficient in FRS2 palmitoylation can proliferate, FGF1-induced neuronal differentiation strictly depends on the palmitoylation of the docking protein.

biochemistry↗

bioRxiv2025

AI-Guided Stability Tuning of a Heterodimeric Linker for Programmable Protein Tube Architectures

Advances in artificial protein assembly design have enabled the construction of increasingly complex higher-order protein architectures. However, rationally tuning the morphology of such assemblies beyond their initially formed architectures remains challenging. Here, we show that the thermal responsiveness of a heterodimeric coiled-coil linker provides a molecular handle for tuning the formation and morphology of two-component protein tubes. Guided by computational stability predictions, we generated a panel of single-amino-acid variants in the M3L2/p66 linker and identified assembly-competent variants with distinct, experimentally accessible thermal transition behaviors. Incorporating two such variants into the tube-forming scaffold, together with the wild-type linker, enabled a focused comparison across three linker states, revealing stepwise shifts in tube-forming temperature windows and tube diameters. Notably, the variant with the lowest apparent thermal stability in this comparison uniquely accessed multi-walled tube architectures. Time-course electron microscopy of this variant revealed a sequential pathway from initially formed thin tubes to wall-thickened intermediates and ultimately to multi-walled states. Together, these findings establish linker thermal responsiveness as a design handle for tuning protein nanotube morphology and expanding the accessible structural states of designed protein tubes.

bioengineering↗

bioRxiv2025

Csf1, a tunnel-like lipid-transfer protein, mediates lipid remodeling and underpins eukaryotic membrane resilience to high hydrostatic pressure and cold

Biological membranes continuously remodel their lipid composition to preserve functionality under environmental stress, yet the molecular basis of this process in eukaryotes remains incompletely understood. Here, we identify the tunnel-like lipid transfer protein Csf1 as a central factor mediating adaptive lipid remodeling that enables Saccharomyces cerevisiae to tolerate high hydrostatic pressure and low temperature. Quantitative lipidomic and membrane biophysical analyses revealed that loss of Csf1 markedly reduces the unsaturation of phosphatidylserine (PS) and phosphatidylethanolamine (PE), leading to rigidification of the endoplasmic reticulum (ER) membrane. Whereas OLE1 overexpression partially mitigated this defect at low temperature, no compensatory response occurred under pressure. Overexpression of the PS/phosphatidylinositol 4-phosphate exchanger Osh6/7 restored PS and PE unsaturation and rescued growth of the Csf1-deficient mutant, indicating a cooperative role in sustaining PS flux at ER-plasma membrane (PM) contact sites. Pressure-induced degradation of the amino-acid permease Bap2 further linked lipid imbalance to membrane-protein instability. By supplying unsaturated PS and PE, Csf1 preserves ER and PM flexibility, defining a conserved mechanism of eukaryotic membrane adaptation to extreme physical stress.

cell biology↗

bioRxiv2025

Hox - Meis-relayed topographical genetic switch underlies cardiopharyngeal neural crest diversification, revealed by multimodal analysis

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks (GRNs) underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and GRN architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx GRN defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

developmental biology↗

bioRxiv2025

Spontaneous mutations in braRS and braAB and the IS1181-related deletion of vraDE genes increase the susceptibility of Staphylococcus aureus clinical isolates to epidermin

Bacteriocins, which are antimicrobial factors produced by bacteria, are expected to be candidates for a novel class of antimicrobial agents against antimicrobial-resistant bacteria, including methicillin-resistant Staphylococcus aureus. To evaluate its effectiveness, the examination of susceptibility using multiple clinical isolates is necessary. In this study, we focused on epidermin, a class I bacteriocin produced by Staphylococcus epidermidis. Among the 146 S. aureus clinical isolates, 77% did not show susceptibility to epidermin, but the susceptibility of some of the strains was significantly higher. Knowing that the two-component regulatory system BraRS affects the susceptibility to epidermin, we analyzed the inducibility of the effector gene vraD in susceptible strains and found that its ability is lost in highly susceptible strains. Some of these strains harbor frameshift or nonsense mutations in BraR, BraA and BraB. Interestingly, the vraDE genes with 35,005 bp neighboring genomic regions, including the icaRADBC and histidine biosynthesis operons, were lost in some highly susceptible strains. In addition, all the vraDE-deficient strains were CC121, and IS1181 sequences located immediately upstream and downstream of the deleted region were found in some vraDE-positive CC121 strains. The deleted region was replaced by one copy of the IS1181 sequence in vraDE-deficient strains. Subculturing of the vraDE-positive CC121 strain generated a vraDE-deficient mutant with increased susceptibility to epidermin at a rate of approximately 1%, suggesting that recombination between IS1181 sequences results in the deletion of the genomic region, including vraDE, in some CC121 strains.

microbiology↗

bioRxiv2025

Positional identification of a candidate gene for MALE STERILITY 2 (MS2) by linkage mapping and transcriptomic data in Cryptomeria japonica D. Don

BackgroundJapanese cedar (Cryptomeria japonica D. Don) is a major plantation species in Japan, but its abundant pollen production is a primary cause of seasonal allergic rhinitis (pollinosis). To mitigate this public health issue, the use of male-sterile cultivars has been promoted. Five types of recessive male-sterile mutations (ms1-ms5) have been identified, and the causal genes and mutations for MS1 and MS4 have been elucidated. However, the gene responsible for MS2-type male sterility remains unknown. ResultsWe aimed to identify the candidate gene responsible for MS2-type male sterility using a map-based cloning strategy. High-resolution linkage mapping localized the MS2 locus to a 1.56 cM interval on linkage group 5, corresponding to an 8.64 Mb region of the reference genome. Ninety-one genes in this region were subjected to functional annotation, gene expression analysis, and mutation screening. Among these, a single gene, SUGI_0493010, encoding a GDSL-type esterase/lipase protein (GELP), fulfilled all three criteria: it showed homology to pollen development genes in Arabidopsis thaliana, was specifically expressed in male strobili, and carried a deleterious amino acid substitution (S40F) within the predicted catalytic domain in ms2 mutant. The same mutation was also detected in a heterozygous individual (Ms2/ms2) from a separate breeding population, whose genotype was confirmed through progeny testing. Structural annotation revealed that the affected serine residue lies within the conserved GDSL motif, suggesting a functional disruption of enzymatic activity. ConclusionsOur results strongly suggest that SUGI_0493010 (GELP) is the causal gene for MS2-type male sterility in C. japonica. This finding enhances our understanding of male sterility mechanisms in conifers and provides a valuable genetic resource for breeding pollen-free trees. The study also demonstrates the effectiveness of combining genetic mapping with transcriptomic and mutational data in forest tree genomics.

genomics↗

bioRxiv2025

Kidney-specific lipid metabolism underlies stable chemical individuality in domestic cats and other felids

Domestic cats uniquely accumulate lipid droplets (LDs) in renal proximal tubular epithelial cells, but their chemical composition and biological functions remain unknown. Here, we identify a distinctive renal specialization in felids: lipid droplets enriched in branched-chain fatty acids (BFAs). These BFAs accumulate as triglyceride components and are excreted in urine as free fatty acids with stable and individually distinctive profiles. Behavioral assays show that cats discriminate urine samples based on BFA composition, even against complex volatile backgrounds, suggesting BFAs as robust, metabolically encoded olfactory signatures for individual recognition. Comparative analyses of non-domesticated felids reveal that BFA production is broadly conserved but exhibits both species-specific and individual variation. These findings demonstrate how a core metabolic pathway has been evolutionarily co-opted into a chemical signature system. This discovery highlights an unusual integration of renal physiology with social communication and expand our understanding of lipid metabolism, organ-level adaptation, and the evolution of chemical individuality in mammals. Significance statementUnique lipid droplets in felid kidneys reveal how organ-specific metabolism can be adapted for social communication.

zoology↗

bioRxiv2025

Pathologic profibrotic activation of distinct myeloid cell subsets in a model of impaired healing after anterior cruciate ligament reconstruction

Anterior cruciate ligament reconstruction (ACLR) may fail due to poor bone-to-tendon integration, yet the cellular mechanisms underlying this process remain incompletely understood. Using a murine model of ACLR, we show that host-derived immune cells are the primary drivers of early healing, while tendon graft-derived stromal cells also contribute at the tendon-bone interface. We developed a model of impaired post-ACLR healing by a surgical approach, that led to increased infiltration of immune cells at the tendon-bone interface, particularly CX3CR1+ monocytes/macrophages (MonoMacs). Single-cell RNA sequencing revealed dynamic monocyte-to-macrophage transitions, with an enrichment of fibrotic, mechanosensitive CX3CR1+ MonoMacs in the impaired healing model. These findings suggest that excessive recruitment or functional shifts in host-derived MonoMacs may disrupt normal healing dynamics. Our study identifies CX3CR1+ MonoMacs as a key cell population related to inferior bone-to-tendon healing after ACLR and potential therapeutic targets to improve surgical outcomes.

immunology↗

bioRxiv2025

Transcriptome comparison between the cultured and in vivo Chick Primordial Germ Cells by SMART-seq-based single cell RNA sequencing

Primordial germ cells (PGC), the precursors of the germline, have unique cellular characteristics to undergo long-distance migration to the embryonic gonads and have the potential to differentiate into somatic cells. Among the animal models studying PGC development, the chicken PGCs are an ideal model, since it is a rare model in which long-term PGC cultivation is applicable. Although the cultural applicability of chicken PGC makes it attractive for revealing the PGC character and its developmental processes, some differences from endogenous PGCs are known, such as the remarkable up-regulation of cell proliferation and a lesser ability to reach the gonads. Understanding these differences at the molecular level is crucial. To this end, we first performed SMART-seq-based single-cell RNA sequencing to compare transcriptomes between endogenous PGCs and cultivated PGCs. Our results revealed that PGC cultivation causes a shift from a MYC-dependent to a MYCN-dependent gene regulatory network in PGCs, suggesting that this reprogramming contributes to the acquisition of proliferation ability and stem cell characteristics in cultivated PGCs. Additionally, our results suggest that the MYCN-dependent gene regulatory network increases the risk of somatic differentiation, particularly in neural fate, in cultivated PGCs. In addition, our transcriptome analysis identified new cell populations that show molecular character as intermediate cell states between germline and pluripotent cells from the early embryonic stage. Thus, our study provides fundamental molecular information to understand both the effects of PGC cultivation and the developmental process of chicken PGCs.

developmental biology↗

bioRxiv2025

Extracellular K+ modulates the pore conformations of Cys-loop receptor anion channels

K+ is an essential cation for life, but no eukaryotic membrane protein with a modulatory site for extracellular K+ has been discovered. Here, we report that a Cys-loop receptor, CG12344/DmAlka, expressed in the Drosophila nervous system, is selectively modulated by physiological concentration of extracellular K+. Structural prediction, electrophysiology and phylogenetic analysis of DmAlka revealed the extracellular K+ binding site that mimics the hydrated chemical environment for K+, as observed in K+ channel pore. Furthermore, we found that K+ binding induces a previously unrecognized "mode-switching," altering properties ranging from ligand sensitivity to ion selectivity. Notably, a human glycine receptor variant also exhibited similar mechanisms. Our study reveals a novel regulatory mechanism of Cys-loop receptors that directly links the extracellular K+ signaling to Cl- conductance in animals.

biophysics↗

bioRxiv2025

Cell Morphology and Biophysical Mechanisms-Informed Traction Force Microscopy Using Machine Learning

AO_SCPLOWBSTRACTC_SCPLOWQuantitative evaluation of cell motility is essential for understanding biological functions. Traction force microscopy (TFM) is a method for quantifying cellular traction forces. By performing inverse mathematical analysis of substrate deformations induced by cellular forces, it is possible to estimate the underlying traction forces. Among various approaches for detecting substrate deformations, the most widely used is to track the displacement of fluorescent beads randomly embedded in the substrate. However, it is well known that the accuracy of force estimation deteriorates when the observation density is low. Furthermore, standardized datasets have not been established, as validation settings--such as random force distributions on the substrate, cell size, and bead density--vary across studies. To enable quantitative evaluation, we constructed a dataset based on the biophysical mechanism by which cellular traction forces are transmitted to the substrate through stress fibers, clutch proteins, and focal adhesions. In addition, we proposed a novel machine learning model that incorporates cell shape information obtained from observations, and we designed a loss function that accounts for both force magnitude and direction. As a result, our method enables highly accurate force estimation even under sparse observation conditions.

biophysics↗

bioRxiv2025

Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

Mitochondria play a central role in cellular energy metabolism and homeostasis, and their dysfunction is closely linked to the progression of age-related diseases. The mitochondrial ubiquitin ligase MITOL (also known as MARCHF5) is a key regulator of mitochondrial dynamics and function, and reduced MITOL expression in the mouse heart has been implicated in mitochondrial dysfunction and cardiac aging. In this study, we identified berberrubine as a compound that promotes MITOL expression and activates mitochondria. We further assembled a group of berberrubine-based compounds, including its quinoid form and a newly developed water-soluble derivative, and collectively named them "Mitorubin" as mitochondria-activating compounds with therapeutic potential. While conventional berberrubine has poor water solubility, the addition of acetic acid significantly improved its solubility, enabling formulation as a solution. Mitorubin enhanced MITOL expression in cultured cells, increased mitochondrial DNA content and expression of mitochondrial proteins, and promoted mitochondrial respiration. In a model of age-related cardiac dysfunction, oral administration of Mitorubin restored mitochondrial function, improved cardiac performance, suppressed myocardial hypertrophy, and alleviated pulmonary congestion. Moreover, Mitorubin did not shorten lifespan in aged mice and significantly extended lifespan in high-fat diet-fed mice, suggesting both safety and efficacy under chronic administration. These findings suggest that Mitorubin is a promising mitochondrial activator and may represent a novel therapeutic strategy for age-related diseases.

biochemistry↗

bioRxiv2025

Muc6-expressing gastric isthmus progenitors contribute to regeneration and metaplasia supported by myeloid-mesenchymal interactions

Gastric mucosal homeostasis is maintained by tissue-resident stem and progenitor cells residing in the isthmus region. Following mucosal injury, surviving cells contribute to regeneration, coinciding with characteristic pathological changes such as atrophic gastritis and metaplasia. To comprehensively understand the cellular dynamics involved in this process, we performed single-cell and spatial transcriptomics using newly generated transgenic mice. In human samples and mouse models, loss of gastric chief cells precedes, and even induces, loss of parietal cells during the progression of atrophy and metaplasia, validating the causal relationship underlying the decrease of these two lineages. Single-cell analysis confirmed robust stemness and metaplastic changes in the Muc6-expressing neck lineage following either chief or parietal cell ablation, and lineage-tracing experiments revealed that Muc6-expressing isthmus progenitors serve as a source of metaplasia and regeneration. Mechanistically, mucosal injury recruits IL-1-expressing myeloid cells, which stimulates NRG1 production in stromal fibroblasts, leading to mucosal proliferation and regeneration mediated by Myc activation in isthmus progenitors. These findings highlight the injury-responsible stem cell-like function of Muc6-expressing isthmal progenitors, which play a critical role in mucosal homeostasis and disease progression. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/648856v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@10fc2eaorg.highwire.dtl.DTLVardef@1c5b82corg.highwire.dtl.DTLVardef@1be747dorg.highwire.dtl.DTLVardef@d1d722_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

The Masc-PSI complex directly induces male-type doublesex splicing in silkworms

The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori, a lepidopteran model insect, females carry the W chromosome with the feminizing gene Feminizer (Fem), which is the source of a female-specific PIWI-interacting RNA (piRNA). The Fem piRNA-PIWI complex cleaves an mRNA encoding the masculinizing protein Masculinizer (BmMasc), resulting in the production of the female-type splice variant of Bombyx mori doublesex (Bmdsx), which is the master genetic switch of B. mori sex differentiation. In contrast, in males, BmMasc induces the production of the male-type Bmdsx splice variant (BmdsxM). However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the male-specific Bmdsx splicing event remains unknown. In this study, we showed that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), that is a RNA binding protein required for BmdsxM expression. BmMasc overexpression resulted in the production of BmdsxM in B. mori ovary-derived BmN-4 cells, but this induction was severely inhibited in BmPSI-knocked down cells, indicating that BmPSI is essential for the masculinizing activity of BmMasc. We also identified that BmMasc-containing protein complex was associated with a region of Bmdsx pre-mRNA spanning introns 2 to 4, particularly around the junctions of intron 2 with exon 3 and intron 3 with exon 4. Exons 3 and 4 of Bmdsx are female-specific units and are skipped in BmdsxM. Together with a previous report of the binding of BmPSI to the CE1 sequence located in exon 4 of Bmdsx, the current results strongly suggest that the BmMasc-BmPSI complex is required for male-specific exon skipping in Bmdsx pre-mRNA through its binding to the female-specific Bmdsx introns and exons.

molecular biology↗