Biology subjects

Masuda, T.

Publications and source records attributed to Masuda, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of Novel Gamma Radiation-Responsive Genes in the Thiosulfate-Oxidizing Bacterium Limnobacter thiooxidans

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

microbiology↗

bioRxiv2026

An organotypic neocortical slice culture for studying neuroglial interactions

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

neuroscience↗

bioRxiv2026

The strictly conserved GGG-tracts in the 5' and 3' long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

Three consecutive deoxyguanosine residues (the GGG-tract) in the U3/R junction of the 5 long terminal repeat (LTR) are strictly conserved among all HIV-1 subtypes. Each deoxyguanosine within the tract has been reported to function as transcription initiation site for HIV-1 RNA. Furthermore, RNAs whose transcription initiates from the third deoxyguanosine in the tract (1G form RNAs) are predominant in virus particles and serve as the primary templates for reverse-transcription. In this study, we generated mutant HIV-1s by replacing the tracts in both the 5 and 3 LTR with other nucleotides to elucidate their functional significance. We identified several proviral sequences containing unexpected mutations near the 5 tract after infection with the mutant, but not the wild-type virus. Five-prime rapid amplification of cDNA end (5 RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5 terminal sequences differing from the plasmid used for producing the particles. Some of the unexpected proviral sequences likely arise directly from these variants during reverse-transcription. We also found that replacing all three nucleotides in the 3 tract with deoxyadenosines decreased the proportion of the 1G form RNAs in particles to 32.6%. Nevertheless, up to 88% of provirus was likely generated with the 1G form RNAs, although they were no longer absolutely predominant in particles. Our results demonstrate that the GGG-tracts in the 5 and 3 LTR are conserved to maintain the integrity of reverse-transcription for LTR sequence generation by controlling multiple steps of HIV-1 replication, including transcription, RNA packaging and reverse-transcription. ImportanceHIV-1 is highly mutable, yet certain conserved sequences remain consistent across most strains. These sequences are thought to be maintained because mutations in these regions typically impair viral fitness, leading to the elimination of such variants through natural selections. This study suggests that HIV-1 has a unique mechanism to autonomously prevent acquisition of mutations in certain regions. Specifically, the GGG-tracts in the 5 and 3 LTR ensure accurate transcription of HIV-1 RNAs, selective packaging of the 1G form RNAs, and preferential use of these 1G forms as a template for reverse-transcription. GGG preservation minimizes unwanted mutations in this region, allowing progeny virus to inherit intact LTR sequences. Interestingly, the preferential usage of 1G form RNAs as template for reverse-transcription is not due to their predominance in virus particles. This suggests that genome packaging independently contributes to reverse-transcription as preparatory stage by concentrating the most suitable RNAs for reverse-transcription in particles.

microbiology↗

bioRxiv2026

Vicious circle of amyloid and leptomeningeal macrophages evokes vascular dysfunction in CAA

The subarachnoid space contains leptomeningeal arteries and resident immune cells known as subarachnoid macrophages (SAMs). In Alzheimers disease (AD) and related cerebral amyloid angiopathy, amyloid-{beta} (A{beta}) frequently accumulates around the leptomeningeal arteries. Nevertheless, the local immune responses and their functional consequences remain poorly understood. Using longitudinal intravital imaging in a mouse model of AD, we tracked A{beta} deposition and identified a distinct SAM population that migrates to and phagocytoses these arterial A{beta} deposits. SAM recruitment correlated with vascular remodeling, including smooth muscle loss, aneurysm formation, and reduced cerebral perfusion. Transcriptomic profiling revealed a distinct SAM population in AD with upregulated CD39 expression. Pharmacological inhibition of CD39 attenuated arterial A{beta} deposition, identifying SAMs as a potential therapeutic target in AD.

neuroscience↗

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↗

bioRxiv2026

Csf1r-mediated depletion of midbrain microglia prevents dopaminergic neuron loss during chronic colitis

Inflammatory bowel disease (IBD) predisposes to neuropsychiatric comorbidity and particularly increases the risk of Parkinsons Disease (PD) in later life. Although the gut-immune-brain axis was proposed as a link between IBD and PD and a driver of PD immunopathogenesis, the regional pattern and single-cell landscape of the brain immune response during colitis and its contribution to PD pathology remain poorly defined. Here, we observe a loss of dopaminergic neurons in the substantia nigra pars compacta of adult mice with chronic colitis. By confocal microscopy and integrated multi-omics, we reveal a complex midbrain-centered immune response to chronic colitis in comparison to the cortex, hippocampus, and striatum. Single-cell mapping of the midbrain immune landscape showed an inflammatory shift of microglial clusters including an expansion of interferon-response microglia, CD8+ T cell extravasation, and increased numbers of vessel-associated neutrophils. Selective myeloid cell depletion using a colony stimulating factor 1 receptor (Csf1r) inhibitor after colitis onset reduced midbrain microglia by 67% and led to a complete rescue of dopaminergic neuron loss, without affecting mucosal pathology or T cell and neutrophil migration to the midbrain. Collectively, within the complex innate and adaptive midbrain immune response to chronic colitis, we demonstrate a causal role of Csf1r-dependent microglia for dopaminergic neurodegeneration. Thus, Csf1r inhibition in IBD may not locally ameliorate colitis, but provide neuroprotection to dopaminergic neurons. These results reveal a novel cellular link between chronic gut-derived peripheral inflammation and midbrain vulnerability and thereby substantially enhance our understanding of the risk for PD related to the gut-immune-brain axis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/700559v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1bf7217org.highwire.dtl.DTLVardef@1db66c0org.highwire.dtl.DTLVardef@136ef2eorg.highwire.dtl.DTLVardef@190f641_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. C_FIG

neuroscience↗

bioRxiv2025

Introduction of regioselective bacterial heme oxygenases into Arabidopsis hy1-1 supports the retrograde heme signaling hypothesis

Heme is synthesized in the plastid and catabolized by heme oxygenase (HO). In Arabidopsis, HO1/HY1/GUN2 predominantly functions for heme catabolism. Previously, we demonstrated that introduction of either plastid-or cytosol-localized HO1 into gun2 or hy1-1 complemented the phenotypes including genomes uncoupling (gun), indicating the assembly of functional phytochromes, as well as supporting the heme signaling hypothesis. To dissect the heme signaling and phytochrome assembly, in this study, we introduced either of the two types of regiospecific bacterial HOs that produce biliverdin IX (BVIX) or BVIX{beta}/{delta} into hy1-1. Introduction of BVIX-producing HO complemented the long hypocotyl, low pigmentation, and gun phenotypes of hy1-1. Interestingly, the introduction of BVIX{beta}/{delta}-producing HO failed to complement the long hypocotyl and low pigmentation phenotypes, suggesting failure of functional phytochrome assembly. However, these lines restored the gun phenotype, thus supporting the heme signaling hypothesis. Based on the levels of complementation of the gun phenotype, we found that the expression of photosynthesis-associated nuclear genes (PhANGs) can be separated into phytochrome-dependent and -independent groups. Our results demonstrate that heme functions as a retrograde mobile biogenic signal from plastids, passing through the cytosol, to regulate the expression of PhANGs, and this regulation is distinct in its dependency of phytochromes.

plant biology↗

bioRxiv2025

Genome-edited retinal organoids restore host bipolar connectivity in the primate macula

Retinal organoids (ROs) represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but whether host cone bipolar cells (BCs) in the primate macula can rewire with transplanted photoreceptors remains unresolved. Here, we transplanted genome-edited human retinal organoids lacking ON-BCs (Islet-1-/- ROs) into a non-human primate macular degeneration model. Remarkably, host rod and cone BCs extended dendrites toward grafted photoreceptors, forming functional synapses confirmed by immunohistochemistry, ultrastructural imaging, and focal macular electroretinography. Both ON- and OFF-pathway connectivity was rebuilt, providing the first demonstration of host-graft synaptic integration in the primate macula. These results establish that primate cone circuits retain a surprising capacity for rewiring and highlight genome-edited ROs as a powerful platform for vision restoration. Our findings represent a critical translational step toward stem cell-based therapies capable of repairing central vision in patients with advanced macular degeneration.

neuroscience↗

bioRxiv2025

Inflammation-enhanced synapse-specific phagocytosis by adult APP microglia in a microfluidic neuron-microglia co-culture model

Microglia play a critical role in synapse remodeling and neuroinflammation, both of which are dysregulated in Alzheimers disease (AD). However, most in vitro models rely on neonatal or immortalized microglia, limiting their relevance to adult pathophysiological context. Here, we present a compartmentalized microfluidic co-culture platform that enables spatially controlled interactions between primary cortical neurons and adult microglia from wild-type (WT) and APP-transgenic mice. This system allows precise functional analysis of microglia-synapse interactions under defined inflammatory conditions. Upon lipopolysaccharide (LPS) stimulation, APP microglia exhibited exaggerated morphological activation, elevated IL-1{beta} secretion, and selectively increased engulfment of synaptic material. In contrast, phagocytosis of non-specific substrates such as pHrodo Zymosan remained unchanged, suggesting a substrate-specific enhancement of microglial phagocytic activity. Blocking the complement receptor CD11b abolished the LPS-induced increase in synaptic uptake, confirming the role of complement-dependent pathways. Transcriptomic profiling revealed robust inflammatory responses in both genotypes, with selectively heightened expression of proinflammatory genes in APP microglia, consistent with a primed immune phenotype. Importantly, increased synaptic uptake occurred without measurable loss of global synaptic connectivity, highlighting the specificity and sensitivity of the system to detect microglial functional changes. This model captures genotype-dependent microglial reactivity (revealing phenotypes not fully captured by transcriptomic rofiling) and provides a physiologically relevant, tractable in vitro platform for dissecting microglial contributions to synaptic pathology in neurodegenerative disease.

neuroscience↗

bioRxiv2025

Genomic characterization of sub-populations in human pluripotent stem cell-derived retinal progenitor cells that drive retinal layer structure

The mechanism underlying retinal spheroid layer formation was investigated using Rax::GFP-positive retinal progenitor cells from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing revealed that well-layered spheroids transiently activated canonical WNT2B-FZD7 signaling followed by temporary expression of non-canonical WNT5A. Despite structural differences in vitro, non- layered and well-layered retinal spheroids on differentiation Day 60 successfully developed into cone and rod subtypes of photoreceptors and established synaptic connections with host bipolar cells after transplantation in a retinal degeneration rat model, resulting in light-evoked functional recovery. Additionally, part of the presumable Rax::GFP-positive retinal progenitor cells differentiated into non- retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium, and spinal cord-like tissues in vitro, reflecting the developmental plasticity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, whereas environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.

cell biology↗

bioRxiv2025

SLFN11-mediated tRNA regulation induces cell death by disrupting proteostasis in response to DNA-damaging agents

DNA-damaging agents (DDAs) have long been used in cancer therapy. However, the precise mechanisms by which DDAs induce cell death are not fully understood and drug resistance remains a major clinical challenge. Schlafen 11 (SLFN11) was identified as the gene most strongly correlated with the sensitivity to DDAs based on mRNA expression levels. SLFN11 sensitizes cancer cells to DDAs by cleaving and downregulating tRNALeu(TAA). Elucidating the detailed mechanism by which SLFN11 induces cell death is expected to provide insights into overcoming drug resistance. Here, we show that, upon administration of DDAs, SLFN11 cleaves tRNALeu(TAA), leading to ER stress and subsequent cell death regulated by inositol-requiring enzyme 1 alpha (IRE1). These responses were significantly alleviated by SLFN11 knockout or transfection of tRNALeu(TAA). Our proteomic analysis suggests that tRNALeu(TAA) influences proteins essential for maintaining proteostasis, especially those involved in ubiquitin-dependent proteolysis. Additionally, we identified the cleavage sites of tRNALeu(TAA) generated by SLFN11 in cells, and revealed that tRNA fragments contribute to ER stress and cell death. These findings suggest that SLFN11 plays a crucial role in proteostasis by regulating tRNAs, and thus determines cell fate under DDA treatment. Consequently, targeting SLFN11-mediated tRNA regulation could offer a novel approach to improve cancer therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/632070v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@9a9e27org.highwire.dtl.DTLVardef@1053186org.highwire.dtl.DTLVardef@1f67ae4org.highwire.dtl.DTLVardef@fe3091_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Interleukin-4 induces CD11c+ microglia leading to amelioration of neuropathic pain in mice

Neuropathic pain, a debilitating chronic pain condition, is a major clinical challenge. The pleiotropic cytokine interleukin-4 (IL-4) has been shown to suppress neuropathic pain in rodent models, but its underlying mechanism remains unclear. Here, we show that intrathecal administration of IL-4 to mice with spinal nerve transection (SpNT) increased the number of CD11c+ microglia (a microglia subset important for pain remission) in the spinal dorsal horn (SDH) and that this effect of IL-4 was essential for its ameliorating effect on SpNT-induced pain hypersensitivity. Furthermore, in mice with spared nerve injury (SNI), another model in which pain remission does not occur, the emergence of CD11c+ SDH microglia was curtailed, but intrathecal IL-4 increased their emergence and ameliorated pain hypersensitivity in a CD11c+ microglia-dependent manner. Our study reveals a mechanism by which intrathecal IL-4 ameliorates pain hypersensitivity after nerve injury and provides evidence that IL-4 increases CD11c+ microglia with a function that ameliorates neuropathic pain.

pharmacology and toxicology↗

bioRxiv2024

Single-cell, spatial, and fate-mapping analyses uncover niche dependent diversity of cochlear myeloid cells.

Recent advances in fate mapping and single-cell technologies have revealed how the dynamics and function of tissue-resident macrophages are shaped by their environment. However, macrophages in sensory organs such as the cochlea where the central nervous system and peripheral nervous system meet remain understudied. Combining single-cell transcriptomics, fate mapping, and parabiosis experiments, we show that five types of myeloid cells including three tissue-resident macrophage subpopulations, coexist in the mouse cochlea. The three macrophage subsets showed different potential functions in relationship with their specific topography across cochlear compartments. Further analysis revealed that they were partially derived from yolk sac progenitors during development, while in adulthood, most cochlear macrophages were long-term resident. Finally, we showed that cochlear macrophage morphology and density changed during aging. Our findings show that cochlea is a microenvironment with a unique heterogeneity of macrophages in terms of gene expression, spatial distribution, ontogeny, and function.

immunology↗

bioRxiv2024

Evolutionary conserved RLF, a plant cytochrome b5-like heme-binding protein, is essential for organ development in Marchantia polymorpha

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b5-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root formation. Whereas the other Cytb5-HBD proteins in A. thaliana regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function, and whether RLF orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme in vitro and that the two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, MpRLF, a RLF ortholog in Marchantia polymorpha, rescues the lateral root formation phenotype of the A. thaliana rlf mutant. Mprlfge, the loss-of-function mutation in the MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and antheridiophore formation. Transcriptome analysis using Mprlfge revealed that MpRLF affects several metabolic pathways. Our findings indicate that MpRLF is essential for vegetative and reproductive development in M. polymorpha, suggesting that the RLF-dependent redox reaction systems are evolutionarily conserved as crucial mechanisms for organ development across diverse plant species.

plant biology↗

bioRxiv2024

Advancing species identification: A non-invasive molecular approach through spider silk proteome analysis

Species identification is crucial in various scientific disciplines such as biology, ecology, medicine, and agriculture. While traditional methods rely on morphological characteristics, DNA barcoding has gained popularity due to its molecular biology approach. Nonetheless, DNA barcoding can be problematic for small animals such as insects, as it requires damaging their bodies for DNA extraction, impacting subsequent breeding and experiments. In this paper, we propose a non-invasive molecular method for species identification that examines the protein composition of animal produced biomaterials. We chose spider silk, with species-specific protein sequences, as our subject of analysis. First, we established a universal silk-dissolving method that applies to silks from various species. We constructed a bioinformatics pipeline employing metrics of significant difference through proteomic analysis to identify spider species by analyzing peptide sequences present in silk proteins. As a result, we achieved a species identification accuracy of 86% across15 species. An appropriate reference dataset was successfully created, in addition, we also discovered some species are difficult to distinguish due to sequence similarities. This technology has been confirmed to be applicable to spider webs taken from the field. This non-invasive approach can complement DNA barcoding, especially in situations where it is infeasible, such as in studies involving spider-parasitoid wasps that eat spiders. Furthermore, it can be applied to other organisms that release biological substances, such as silkworm pupae, termite digestive enzymes, and tick saliva, aiding in species identification and pest control efforts.

molecular biology↗

bioRxiv2024

Arabidopsis GENOMES UNCOUPLED PROTEIN1 binds to plastid RNAs and promotes their maturation

Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development. GENOMES UNCOUPLED1 (GUN1) plays a central role in retrograde signaling during early plant development. The putative function of GUN1 has been extensively studied, but its molecular function remains controversial. Here, we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling relevant conditions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling. Our study of the plastid (post)-transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome. By combining this result with a PPR code-based prediction and experimental validation by RNA immunoprecipitation experiments, several targets of GUN1 were identified, including 23S rRNA, tRNAs and RNAs derived from ycf1.2 and the ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD gene cluster. The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants account for the cotyledon phenotype. Our study identifies RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies. We anticipate that our findings will serve as a basis for subsequent studies investigating the mechanism of plastid gene expression and will facilitate the elucidation of GUN1s function in retrograde signaling.

plant biology↗

bioRxiv2023

Cytosolic heme metabolism by alternative localization of heme oxygenase 1 in plant cells

Heme, an organometallic molecule, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. Additionally, heme serves as a precursor to phytochromobilin, the chromophore of plant phytochrome. Heme oxygenase (HO) initiates the first committed step in heme metabolism. Our transcription start site-sequencing (TSS-seq) revealed that HO1 in Arabidopsis thaliana and Oryza sativa (rice) has two TSSs, producing long (HO1L) and short (HO1S) transcripts, with or without an intact N-terminal plastid transit peptide. HO1L and HO1S products localize in plastids and the cytosol, respectively. In Arabidopsis, HO1L is prevalent in light-exposed shoots, while HO1S is clearly detected in roots and etiolated seedlings. During de-etiolation and early development, HO1L ratio gradually rises and HO1S ratio decreases. Light perception via phytochrome and cryptochrome elevates HO1L ratio and reduce HO1S ratio through the functioning of HY5 and HYH transcription factors, and the suppression of DET1, E3 ubiquitin ligase COP1, and PIFs transcription factors. As expected, HO1L product was able to complement the HO1-deficient mutant gun2 (hy1), but surprisingly, HO1S expression could also restore the short hypocotyl phenotype and high pigment content, and make the mutant recover from the gun phenotype. This indicates the formation of functional holo-phytochrome within these lines. Our work highlights the presence of a cytosolic pathway for heme metabolism, especially during etiolation and early development. Furthermore, it supports the hypothesis that a mobile heme signal is involved in the mediation of retrograde signaling from the chloroplast. Significance StatementIn this research, through both TSS-seq and CAGE-seq, we discovered that the HO1 (GUN2 or HY1) gene in both Arabidopsis and rice has two TSSs, generating HO1L and HO1S transcripts. We reveal that the HO1 TSS regulation pathway is the same as the light signaling pathway. Significantly, our study identifies that a cytosolic heme metabolism pathway is existent in plant cells.

plant biology↗

bioRxiv2023

Mitochondrial translation regulates terminal erythroid differentiation by maintaining iron homeostasis

A lack of the mitochondrial tRNA taurine modifications mediated by mitochondrial tRNA translation optimization 1 (Mto1) was recently shown to induce proteostress in embryonic stem cells. Since erythroid precursors actively synthesize the hemoglobin protein, we hypothesized that Mto1 dysfunctions may result in defective erythropoiesis. Hematopoietic-specific Mto1 conditional knockout (cKO) mice were embryonic lethal due to niche-independent defective terminal erythroid differentiation. Mechanistically, mitochondrial oxidative phosphorylation complex-I was severely defective in the Mto1 cKO fetal liver and this was followed by cytoplasmic iron accumulation. Overloaded cytoplasmic iron promoted heme biosynthesis and enhanced the expression of embryonic hemoglobin proteins, which induced an unfolded protein response via the IRE1-Xbp1 signaling pathway in Mto1 cKO erythroblasts. An iron chelator rescued erythroid terminal differentiation in the Mto1 cKO fetal liver in vitro. The new point of view provided by this novel non-energy-related molecular mechanism may lead to a breakthrough in mitochondrial research.

cell biology↗