Biology subjects

Kosako, H.

Publications and source records attributed to Kosako, H..

At least 19 recordsLinked to original sources

bioRxiv2026

2'-O-Methylated ncRNA condensates form a recombination center for IgH synapsis

How fibrillarin (FBL), an RNA 2'-O-methyltransferase, regulates antibody diversification remains unclear. This study demonstrated that FBL promotes switch (S)-S DNA synapsis at the IgH locus during class switch recombination (CSR), without affecting DNA cleavage. Both RNA-binding and catalytic activities of FBL were essential for CSR. Moreover, MED19 supported DNA cleavage and synapsis by regulating snoRNAs that guide 2'-O-methylation. Mechanistically, FBL methylated -germline transcripts (-GLTs), cis-acting noncoding RNAs forming nuclear condensates harboring machinery for RNA processing, RNP remodeling, and chromatin dynamics. FBL-mediated 2'-O-methylation governed condensate protein composition. FBL suppression aberrantly enriched nucleolar proteins, including WDR74. Crucially, low WDR74 expression in B cells promoted - and -GLT condensate fusion, thereby enhancing S-S synapsis, CSR, and IgH/c-Myc translocation, potentially contributing to poor myeloma prognosis. Altogether, our findings revealed that RNA 2'-O-methylation dictates GLT condensate composition and fusion dynamics to establish a recombination center, driving genomic DNA synapsis in B cells.

molecular biology↗

bioRxiv2026

Androgen receptor determines skeletal muscle sexual dimorphism

The molecular and functional bases of sexual dimorphism in skeletal muscle remain poorly understood. The androgen receptor (AR) is a major regulator of sex-biased gene expression in muscle, but its genomic targets and associated coregulators in vivo are incompletely defined. Using ChIL-seq and an AirID-AR knock-in mouse, we mapped AR-bound genes and AR-associated proteins in skeletal muscle and identified histone deacetylase-linked corepressors. We further identified myosin binding protein H (Mybph) as a female-biased AR-repressed gene conserved in mouse and human muscle. Mybph loss disrupted sarcomeric organization and selectively delayed postinjury force recovery in female mice. These findings define an in vivo AR regulatory network and identify AR-dependent Mybph repression as a potential mechanism contributing to skeletal muscle sexual dimorphism.

molecular biology↗

bioRxiv2026

Oncogenic NPM-ALK reprograms the TGM1 1 interactome toward oncogenic signaling and transcriptional states

Oncogenic NPM-ALK drives aberrant signaling networks that promote malignant phenotypes; however, the molecular mechanisms linking oncogenic signaling to downstream cellular programs remain incompletely understood. Among candidate regulatory factors, transglutaminase 1 (TGM1) has not been functionally characterized in this context. Here, we investigated the role of TGM1 in NPM-ALK-expressing cells by combining proximity-dependent proteomics with functional analyses. Using a TurboID- based approach, we mapped the TGM1-associated protein network and identified extensive remodeling of this network upon NPM-ALK expression. Proteomic analyses revealed that NPM-ALK reduced TGM1-associated proteins involved in genome maintenance and DNA repair, while enhancing associations with proteins linked to cytoplasmic translation and PI3K-AKT signaling pathways. Consistent with these findings, TGM1 deficiency impaired cell proliferation without significantly affecting cell viability, indicating a specific role in maximal proliferative capacity. Furthermore, proteomic and functional analyses suggested a link between TGM1 and AKT signaling pathways. Together, these findings suggest that oncogenic NPM-ALK reprograms the TGM1 interactome toward oncogenic signaling and transcriptional states, positioning TGM1 within signaling networks associated with proliferative cellular phenotypes.

molecular biology↗

bioRxiv2026

Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

Understanding how molecular diversity across long-range neural circuits governs brain function remains a central challenge in neuroscience. Here, we developed a projection-specific spatial proteomics approach and revealed robust presynaptic molecular divergence across six projection-defined pathways of the medial prefrontal cortex (mPFC), including efferent projections to the basolateral amygdala (BLA), nucleus accumbens (NAc), thalamus (Thal), hypothalamus (HT) and cortex (CTX), as well as the afferent projection from the BLA to the mPFC. Among these pathways, we identify BLTP2 (KIAA0100), a previously uncharacterized transmembrane protein, as highly enriched in the projection from the mPFC to the BLA. BLTP2 localizes to excitatory presynaptic terminals and is enriched at synapses that are activated during memory formation. Loss of BLTP2 impairs synaptic structure and transmission, and reduces activity-dependent remodelling, resulting in selective deficits in contextual fear memory, anxiety-related behavior, and social behavior. Mechanistically, BLTP2 promotes presynaptic assembly by recruiting Neurexin 1. These findings reveal projection-specific presynaptic molecular diversity and provide mechanistic insights into circuit-level vulnerabilities in neuropsychiatric disorders.

neuroscience↗

bioRxiv2025

IDR-dependent MORC1 bodies limit chromatin accessibility to tune transposon repression

Germline-specific MORC1-mediated transposon repression intersects with the MIWI2-piRNA pathway and involves H3K9me3 deposition, yet the underlying mechanism remains unclear. Here, we show that MORC1 assembles nuclear bodies in gonocytes. Using a doxycycline-inducible MORC1 expression system in NIH3T3 cells, we find that condensate formation is confined to the nucleus via an IDR containing a bipartite NLS, and that TRIM28, a corepressor that recruits the H3K9 methyltransferase SETDB1, is enriched in these bodies through protein-protein interactions. An IDR-deficient MORC1 mutant fails to form nuclear condensates but retains TRIM28 binding. ChIP-seq analysis reveals that this mutant exhibits higher occupancy at heterochromatin-embedded transposons than wild-type MORC1, with a pronounced preference for evolutionarily young transposon subfamilies that strongly overlap endogenous MORC1 targets in gonocytes. Wild-type MORC1 shows weaker enrichment at these elements and instead exhibits a greater tendency to occupy open chromatin. Condensates formed by exogenous MORC1 expression in NIH3T3 cells are substantially larger than those observed in gonocytes. These findings support a model in which MORC1-dependent transposon repression is governed by physical engagement with TRIM28 and by IDR-dependent, yet quantitatively restrained, nuclear body formation, which limits MORC1 chromatin accessibility, curtails off-target binding, and concentrates repression on bona fide transposon targets.

molecular biology↗

bioRxiv2025

LRBA promotes drug-induced liver injury and MASLD by scaffolding MAPK activation

LPS-responsive beige-like anchor protein (LRBA) regulates vesicular trafficking and receptor recycling, and its deficiency results in immunodeficiency characterized by hypogammaglobulinemia and autoimmune syndrome. However, its role in liver pathophysiology remains unclear. Here, we reveal a previously unrecognized function of LRBA as a critical intracellular scaffold for mitogen-activated protein kinase (MAPK) activation that promotes liver injury. Lrba-/-mice exhibit reduced acetaminophen (APAP)-induced hepatic necrosis through the suppression of JNK activation. In a model of metabolic dysfunction-associated steatotic liver disease (MASLD) induced by a high-fat, high-cholesterol (HFHC) diet, Lrba deficiency reduces hepatic inflammation, fibrosis, and Kupffer cell activation. Mechanistically, LRBA homodimers directly interact with specific mitogen-activated protein kinase kinase kinases (MAP3Ks), including transforming growth factor-{beta}-activated kinase 1 (TAK1) and mixed-lineage kinase 3 (MLK3), to facilitate their activation. LRBA, which is primarily expressed in hepatocytes under physiological conditions, is upregulated in non-parenchymal cells such as Kupffer cells and cholangiocytes in both HFHC diet-fed mice and patients with MASLD and cirrhosis, linking its scaffolding function to pathological inflammation. Thus, LRBA promotes liver disease progression by amplifying inflammatory signaling.

cell biology↗

bioRxiv2025

Sex-biased transcriptome in embryonic mouse cortices under Pax6 haploinsufficiency highlights Pbdc1 as a candidate regulator

Mutations in Pax6, encoding a transcription factor essential for brain patterning and neurogenesis, have been linked to female-biased cortical malformations and behavioral abnormalities, yet the molecular basis remains unclear. Here we show that Pax6 haploinsufficiency (Sey/+) produces more pronounced sex-biased alterations in the transcriptomes and cytoarchitecture of embryonic mouse cortices than those in wild-type and homozygous mutants (Sey/Sey). Pbdc1, a previously uncharacterized X-linked gene implicated in autism, is selectively upregulated in Sey/+ females and proximity-dependent protein-protein interaction analysis reveals Pbdc1 interacts with RNA-splicing factors. Moreover, Pbdc1 overexpression reduces intermediate progenitor cells in the developing cortex. ChIP-qPCR further demonstrates Pax6 and BAF occupancy at the Pbdc1 promoter in WT embryos of both sexes and CUT&Tag shows H3K4me3 elevation selectively in Sey/+ females. Our findings indicate that partial loss of Pax6 shapes the embryonic cortical transcriptomes and cytoarchitecture in a sex-dependent manner and highlight Pbdc1 as a candidate regulator of sex-biased corticogenesis.

developmental biology↗

bioRxiv2025

REFEE Shapes ZGA Through Post-Transcriptional Regulation of Repetitive RNAs in Mouse Embryos

During early mammalian embryonic development, repetitive elements such as transposons and duplicated genes are transcriptionally active and play key roles in embryogenesis. MERVL, an endogenous retrovirus, is essential for shaping the early transcriptome, particularly during the transition from totipotency to pluripotency. However, the function of many repetitive elements remains poorly understood. Here, we add a new repetitive player, REFEE, that regulates repetitive RNAs and contributes to totipotency regulation through post-transcriptional mechanisms. REFEE was initially identified as a paralogue of Alyref, a nucleocytoplasmic RNA exporter, transiently expressed during the 2-cell stage of mouse embryogenesis. REFEE preferentially binds and stabilizes MERVL RNA, promoting its export. REFEE knockdown embryos fail to complete zygotic genome activation (ZGA), accompanied by a significant reduction in MERVL RNA levels. REFEE primarily influences transcript levels of ZGA genes, upregulating or downregulating them in a manner correlated with their genomic proximity to MERVL. Proteomic analysis further revealed that REFEE knockdown also decreases protein levels of its RNA targets, indicating an additional layer of post-transcriptional regulation beyond RNA stability. Our findings identify REFEE as an essential post-transcriptional regulator of early embryonic development and highlight the functional interplay between repetitive elements in shaping the zygotic transcriptome.

developmental biology↗

bioRxiv2025

OMA1-mediated cleavage of AIFM1 upon mitochondrial stress and suppression of cell growth through the control of OXPHOS activity

Mitochondrial proteases regulate the dynamic properties of organelle morphology and ensure functional plasticity at the cellular level. The metalloprotease OMA1 mediates constitutive and stress-inducible processing of its mitochondrial substrates, but the number of functionally characterized targets remains limited. Using multiproteomic and biochemical approaches, we demonstrated that the membrane-anchored inner membrane space (IMS) protein AIFM1 serves as a mitochondrial stress-responsive substrate of OMA1. OMA1 cleaves AIFM1 in the IMS under stress conditions with a kinetically slower reaction than that of its conventional substrate, dynamin-like GTPase OPA1. Membrane dislocation of cleaved AIFM1 in mitochondria reduces its binding to subunits of the oxidative phosphorylation machinery, leading to decreased respiratory activity and ultimately impaired cell growth. Mechanistically, we revealed that AIFM1 broadly safeguards the mitochondrial proteome under steady-state conditions by mediating the import of proteins, particularly respiratory complex I subunits, via the TIM23 complex. These findings reveal a previously unrecognized role for OMA1 in integrating mitochondrial stress sensing and cellular energetics by altering the AIFM1 topology.

cell biology↗

bioRxiv2025

Hypomorphic Lig4 gene mutation in mice predisposes to Th1-skewing intestinal inflammation

Dysregulation of DNA double-strand break (DSB) repair leads to adaptive immunodeficiency, whereas the remaining lymphocytes are aberrantly activated and provoke inflammations. However, no model mice were available to consistently manifest inflammation under defective DSB repair. We generated mutant mice carrying a missense mutation p.W447C in the gene encoding DNA ligase IV (LIG4), critical for DSB repair. Lig4W447C/W447C mice showed growth retardation and severe intestinal inflammations under adaptive immunodeficiency. The inflammations were featured by marked infiltration of T helper type 1 (Th1) cells and macrophages and was dependent on lymphocytes. When Ifng was deleted, Th2 and Th17 instead of Th1 cells drove the inflammations. Lig4W447C/W447C mice showed expansion of oligoclonal T cells with T cell receptor repertoire skewed towards more proximal 3 V and 5 J gene segments. Thus, our novel hypomorphic Lig4 mutant mice show that defective DSB repair leads to Th1-dependent intestinal inflammations under severe adaptive immunodeficiency.

immunology↗

bioRxiv2025

In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID

Heterobifunctional molecules, such as proteolysis-targeting and autophagy-targeting chimera, represent new drug concepts. They are composed of two protein binders that can induce proximity interactions between two proteins and protein catalysis. Currently, cereblon (CRBN)- and von Hippel-Lindau (VHL)-binders with thalidomide- and VH032-backbones are widely used as E3 ligase binders. Here we developed a method to validate proteins that interact with heterobifunctional molecules in cells using AirID, a proximity biotinylation enzyme. Interactome of target proteins was validated for six heterobifunctional molecules. ThBD-AirID, a fusion of the thalidomide-binding domain (ThBD) of CRBN and AirID, effectively biotinylated the target proteins. AirID fused to full-length VHL also exhibited highly effective biotinylation. Heterobifunctional molecules with the same target binder but different E3 binders showed different proximity interactome profiles in cells. Analysis using ThBD-AirID revealed a nuclear interaction between androgen receptor and ARV-110. AirID-fused ThBD and VHL could be useful for validating the heterobifunctional molecular interactome in cells.

molecular biology↗

bioRxiv2025

Linear ubiquitination triggers Amph-mediated T-tubule biogenesis

T-tubules are specialized invaginations of the plasma membrane essential for muscle contraction. While their physiological importance is well established, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis through proximity proteomics and RNAi screening in Drosophila. Loss of LUBEL leads to the formation of Amphiphysin (Amph)-positive membrane sheets instead of tubular networks in muscle cells. Mechanistically, the ubiquitin ligase activity of LUBEL, and direct interaction with Amph, a BAR domain protein involved in membrane tubule extension, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into condensates on membranes, facilitating Amph-mediated membrane tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across a broad range of species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover a previously unrecognized role for linear ubiquitination in membrane deformation driven by BAR domain proteins.

cell biology↗

bioRxiv2025

An in vivo fitness gene of Toxoplasma, MIC11, is essential for PLP1-mediated egress from host cells

After invasion and replication, intracellular pathogens must egress from infected host cells. Toxoplasma gondii facilitates this process by permeabilizing host cells by releasing perforin-like protein 1 (PLP1) through induced microneme secretion. However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identified the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen revealed several genes including MIC11 as an essential gene for virulence. Deletion of MIC11 resulted in severe defects in both membrane rupture and egress. Scanning mutagenesis identified functional motifs in MIC11, and mechanistic analyses demonstrated that MIC11 directly associates with PLP1, regulating its activity in membrane disruption. The MIC11 paralogue MIC22 compensated for MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii. The discovery of MIC11 advances the understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

microbiology↗

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↗

bioRxiv2024

The Rubicon-WIPI axis regulates exosome biogenesis during aging

Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.

cell biology↗

bioRxiv2023

Mitochondrial protein FKBP8 captures PDZD8 to form mitochondria-ER contacts

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

cell biology↗

bioRxiv2023

Golgi membrane proteins YIPF3 and YIPF4 regulate turnover of the Golgi apparatus through autophagy

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the autophagosome. Using quantitative proteomic analysis and two Golgiphagy-reporter systems that we developed, we found that five-transmembrane Golgi-resident proteins, YIPF3 and YIPF4, constitute a Golgiphagy receptor. The YIPF3-YIPF4 complex interacts with LC3B, GABARAP, and GABARAPL1 via the LIR motif in YIPF3, whose stability is dependent on YIPF4. Phosphorylation of the LIR in YIPF3 seems to be required for YIPF3-ATG8 interaction. Moreover, expression of the YIPF3 LIR mutant caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays that we established will pave the way for future studies to obtain deeper insights into Golgiphagy.

cell biology↗

bioRxiv2023

Host ZCCHC3 blocks HIV-1 infection and production by a dual mechanism

Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. While anti-human immunodeficiency virus type 1 (HIV-1) host restriction factors have been identified, most of them are antagonized by viral proteins. This has severely hindered their development in anti-HIV-1 therapy. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel anti-HIV-1 factor that is not antagonized by viral proteins. ZCCHC3 suppresses production of HIV-1 and other retroviruses. We show that ZCCHC3 acts by binding to Gag nucleocapsid protein via zinc-finger motifs. This prevents interaction between the Gag nucleocapsid protein and viral genome and results in production of genome-deficient virions. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. Such a dual antiviral mechanism is distinct from that of any other known host restriction factors. Therefore, ZCCHC3 is a novel potential target in anti-HIV-1 therapy.

microbiology↗