Biology subjects

Iwasaki, S.

Publications and source records attributed to Iwasaki, S..

At least 19 recordsLinked to original sources

bioRxiv2026

RNA-dependent RNA amplification as a strategy to perform tRNA sequencing on low-input samples

Transfer RNAs (tRNAs) are key molecules that deliver amino acids to the translating ribosome according to their cognate codon encoded in messenger RNA (mRNA). Due to the modified nature of tRNA nucleotides, accurate tRNA quantification can be tedious, especially when dealing with small sample inputs. Here, we took advantage of an RNA-dependent RNA amplification method using T7 polymerase to quantify tRNA abundance in low biological input. Our method, called T7 High-resolution original RNA (Thor)-tRNA-Seq, showed reproducible and quantitative measurement of low tRNA inputs. Thus, our Thor-tRNA-Seq is a robust and reliable approach for the quantification of tRNA in samples with small and precious biological material.

molecular biology↗

bioRxiv2026

Neuronal stop-codon readthrough is associated with ribosome pausing and alters protein localization in Drosophila

Stop-codon readthrough (RT) diversifies proteomes and is particularly prominent in neurons, suggesting its importance in nervous systems. However, the regulatory logic that specifies neuronal RT and the structural and cellular consequences of the resulting C-terminal protein extensions remain poorly understood. Here we leverage neuron-specific ribosome profiling datasets in Drosophila to construct an in vivo atlas of 163 neuronal RT transcripts. Sequence- based modeling distinguished RT from non-RT transcripts and highlighted an extended post-stop region enriched for stable predicted RNA structures. Beyond these cis-associated features, ribosome-footprint analysis revealed pronounced stop-codon pausing on RT transcripts, accompanied by upstream periodic peaks from the stop codon consistent with ribosome queuing. At the protein level, neuronal RT appended polypeptides enriched with polar residues and intrinsically disordered regions (IDRs). Finally, an in vivo dual-color reporter showed that RT of the RNA-binding protein Bru3 alters localization from the nucleus to cytoplasmic granules. Together, our results suggest that neuronal RT is associated with structured post-stop RNA regions that reshape termination dynamics and can produce IDR-rich C-terminal protein extensions with distinct subcellular localization.

molecular biology↗

bioRxiv2026

Activity-dependent synthesis of translational machinery through dynamic remodeling of ribonucleoprotein granules

Neuronal stimulation drives gene-expression programs supporting long-lasting changes, yet how depolarization reshapes post-transcriptional regulation remains poorly understood. Here, we combined optogenetic stimulation with neuron-specific Ribo-seq and RNA-seq in the Drosophila brain to resolve activity-evoked translational dynamics. Stimulation triggered two temporally ordered programs: a rapid wave to translate synapse remodeling factors and neuropeptides, followed by a dominant delayed program that selectively upregulated translation of protein synthesis machinery, including nearly all cytosolic ribosomal proteins (RPs). Transcripts in the late program are marked by 5 terminal oligopyrimidine (TOP) motifs. TOP mRNAs accumulated in ribonucleoprotein granules at the basal state but are released upon stimulation. We identified La-related protein as a key regulator of the activity-dependent translation of TOP mRNA and memory consolidation. Comparative genomics indicated that TOP enrichment in RP mRNAs is specific to metazoans. These findings suggest that animals acquired activity-dependent TOP-mRNA control to couple neuronal stimulation with adaptation of translational capacity.

neuroscience↗

bioRxiv2026

Ribosome stalling facilitates chloroplast targeting of nuclear-encoded proteins

Ribosomes translate mRNAs with variable elongation rates and frequently undergo transient stalling. Although ribosome stalling is known to regulate protein quality control and translational dynamics, its transcriptome-wide landscape and biological significance in plants remain largely unexplored. Here, using disome profiling in Arabidopsis thaliana, we generated a high-resolution map of ribosome stalling sites across the transcriptome and uncovered a marked enrichment on mRNAs encoding chloroplast-targeted proteins, particularly those involved in photosynthesis. We further found that ribosome stalling preferentially occurs when transit peptides emerge from the ribosome exit tunnel. Functional assays demonstrated that deletion of the stalling region significantly reduces chloroplast targeting efficiency. Together, our findings identify ribosome stalling as a regulatory layer that promotes efficient targeting of nuclear-encoded proteins to chloroplasts.

molecular biology↗

bioRxiv2026

Translational regulation of GAD1 identified by circadian and light-responsive ribosome-bound transcriptome analysis in the mouse hypothalamic suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) serves as the master circadian pacemaker, integrating regulation of clock genes in the SCN has been extensively studied, whether gene expression in the SCN is regulated at the level of mRNA translation has remained largely unexplored. Here, we report the first ribosome-profiling (Ribo-seq) dataset generated from the mouse SCN, enabling a genome-wide assessment of translational regulation in this central circadian clock. Combined time-series Ribo-seq and RNA-seq analyses identified 385 genes that exhibit rhythmic ribosome binding without corresponding oscillations in mRNA abundance, revealing widespread translational regulation in the SCN. Among these, light stimulation induced an approximately twofold increase in ribosome binding of Gad1, which encodes the major GABA-synthetic enzyme Gad67, despite only marginal changes in Gad1 mRNA levels. Importantly, immunohistochemical analyses demonstrated light-dependent accumulation of Gad67 protein selectively in light-activated SCN neurons, establishing that translational regulation of Gad1 gives rise to overt protein-level changes. In contrast, canonical clock genes were regulated predominantly at the transcriptional level and showed little evidence of translational modulation. Our data allow a systematic comparison between transcriptionally and translationally regulated genes, including their relative rhythm-amplitudes and phases, and thereby revealed translational control as a distinct modulatory layer shaping time-of-day-dependent and light-dependent gene expression in the SCN.

neuroscience↗

bioRxiv2025

Altered Motor Gamma Activity and Hemispheric Lateralisation Reflect Perinatal Maturity in Preterm Children during Motor Control

Preterm birth, defined as delivery before 37 weeks of gestation, is associated with differences in neurodevelopment and an increased likelihood of motor challenges that may persist throughout childhood. However, the neurophysiological correlates of these motor differences remain insufficiently understood. Oscillatory activity in the primary motor cortex is closely linked to motor execution, yet motor-related oscillations have rarely been examined in children born preterm. In this cross-sectional observational study, we investigated motor-related gamma oscillations in 5- to 7-year-old children using a child-customised magnetoencephalography system. Nineteen children born full-term and 18 children born preterm of comparable age performed a child-friendly dominant-hand finger movement task. Movement-related gamma activity was quantified from bilateral precentral gyrus regions used as anatomical proxies for the primary motor cortices. The preterm group showed longer mean response times and greater intra-individual response-time variability than the full-term group (P = 0.003 and P = 0.030, respectively). Contralateral gamma power was also lower in the preterm group (full-term: 59.89 {+/-} 28.87%; preterm: 37.97 {+/-} 25.82%; t(35) = 2.43, P = 0.020, Cohen's d = 0.80). A significant Group x Hemisphere interaction (F(1, 35) = 13.36, P < 0.001, partial 2 = 0.276) further indicated differences in the hemispheric organisation of gamma activity between groups. The gamma laterality index was also lower in children born preterm (full-term: 0.24 {+/-} 0.21; preterm: -0.11 {+/-} 0.37; t(26.4) = 3.51, P = 0.002, Cohen's d = 1.17), consistent with reduced contralateral dominance. Across the whole sample, greater contralateral gamma lateralisation was associated with higher scores on the Hand Movements subtest of the Kaufman Assessment Battery for Children ({rho} = 0.40, P = 0.014, false discovery rate-adjusted q = 0.042) and earlier independent walking ({rho} = 0.57, P < 0.001, q = 0.002). The association with independent walking was also observed within the preterm group ({rho} = 0.78, P < 0.001, q = 0.002). These findings suggest that reduced contralateral gamma power and reduced hemispheric lateralisation in children born preterm may reflect differences in the developmental organisation of motor cortical networks. This study provides new insight into motor cortical organisation and its relationship with motor development following preterm birth.

neuroscience↗

bioRxiv2025

Revisiting the in vivo Functions of Coilin in Mice

Cajal bodies (CBs), first identified in the early 20th century, are classic nuclear structures organized primarily by the protein coilin. Although CBs have long been implicated in snRNA modification and maturation, their precise roles in mice remain unclear. Here, we revisit the functions of CBs by using genome editing to generate mice that lack nearly the entire coilin protein, retaining only the first seven amino acids. Mass-spectrometry analyses showed reduced 2'-O-methylation of U2 snRNA in coilin mutant liver; however, these reductions had minimal impact on overall gene expression and pre-mRNA splicing patterns. Additionally, detailed analysis recapitulated previously reported testicular size reductions and newly revealed decreased proportions of high-quality sperm. We also identified novel coilin-containing condensates distinct from genuine CBs in the nuclear pocket of elongating spermatids. Our findings implicate that coilin orchestrates membraneless organelles in various compartments, playing multifunctional roles beyond classical CB formation.

molecular biology↗

bioRxiv2025

CGK733 binds to adenine nucleotide translocator 2 and modulates mitochondrial function and protein translation

Chemical genetics is a powerful strategy for dissecting biological mechanisms, and a crucial step in this approach is to identify the molecular target responsible for a compounds activity. CGK733, a compound with anti-proliferative activity, was once reported as an ATM/ATR inhibitor, although this activity has been debated, and its target and mode of action have remained unclear. Here, we show that CGK733 inhibits cell-cycle progression and global protein translation. Affinity purification identified adenine nucleotide translocator 2 (ANT2) as the primary target. CGK733 blocked ATP export from mitochondria and induced proton leak, thereby shifting ATP production from mitochondrial respiration to glycolysis and perturbing the TCA cycle. These mitochondrial alterations were accompanied by inactivation of the mTOR pathway and mild activation of the integrated stress response, resulting in translational inhibition. Collectively, our findings demonstrate that CGK733 acts mainly through ANT2-dependent mitochondrial modulation, revealing a mechanistic link between mitochondrial bioenergetics and translational control.

biochemistry↗

bioRxiv2025

Predator-driven local convergence fosters global microbial community divergence

Understanding the rules that govern microbial community assembly is essential for predicting ecosystem function. While microbial predators are key biotic agents that shape bacterial communities through predation, yet their ecological consequences have been studied mostly in isolated and in vitro systems1-3. In contrast, large-scale studies of microbial diversity have primarily emphasized abiotic factors as drivers of community assembly 4-7, while the role of microbial predators modulating global microbial divergence and convergence patterns remains largely neglected. Here, we show that bacterivorous protists (predators) exert dual, scale-dependent effects on microbial communities: promoting local convergence by suppressing dominant bacterial taxa, while generating global divergence through species-specific predation effects. By integrating global meta-analyses, controlled field experiments, and reconstructions of natural and synthetic communities, we find that predator identity and prey susceptibility jointly determine convergence outcomes. Communities dominated by predator-resistant taxa exhibit reduced convergence under predation pressure, revealing a predictable trait-based filtering mechanism. This framework reconciles previous contradictory findings3,8-11 and highlights predators as selective, context-dependent agents of microbial biogeography. Predator-driven convergence suggests new opportunities for microbiome engineering 12: targeted use of predators may steer microbial communities toward functional configurations that enhance soil health, disease suppression, carbon cycling, and ecosystem resilience 2,13,14.

ecology↗

bioRxiv2025

Ultra-parallel ribosome profiling platform with RNA-dependent RNA amplification

Translation regulation plays a pivotal role in the diversification of gene expression and the response to intra- and extracellular environmental cues. Ribosome profiling (or Ribo-Seq) serves as a sensitive, quantitative, comprehensive, and data-rich technique to survey ribosome traversal across the cellular transcriptome. However, due to the intricacy of library preparation, applications to low-input and a large number of samples have presented analytic challenges. Here, we developed the semi-automated platform of Ribo-Seq and Disome-Seq, which allowed us to assess the translation status from a vast collection of samples with reduced amounts in a plate format. Through an siRNA-mediated knockdown screen for ribosome-associated proteins, this technique identified factors that (i) mediate regulation via RNA elements such as the TOP motif, (ii) assist efficient ribosome recycling, (iii) support ribosome-associated quality control (RQC), (iv) enhance translation elongation across inhibitory G-quadruplex sequences, and (v) repress mitochondrial translation. The application to human-derived samples revealed that stop codon readthrough and mitochondrial translation deficiency are associated with severe symptoms in COVID-19. Our approach provides a versatile option to investigate the translatome in a highly parallel manner.

molecular biology↗

bioRxiv2025

Sequence grammar and dynamics of subcellular translation revealed by APEX-Ribo-Seq

Local translation at specific subcellular regions is proposed to define appropriate protein destinations and functions. However, our understanding of local translation still remains far from complete due to a lack of versatile analytical tools. Here, we developed a new method, termed APEX-Ribo-Seq, that integrates ribosome profiling (Ribo-Seq) with APEX2-based proximity labeling. We mapped local translation across 14 distinct organelles and identified over 3,000 genes undergoing localized translation, encoding components of complexes that function at these specific sites. RNA language-model predictors captured compartment-associated information beyond protein sequence, and model-derived attribution nominated sequence-sensitive transcript regions for mechanistic follow-up. Gene-set-level RBP enrichment and knockdown experiments separately identified RBPs associated with compartment-specific translation. The utility of APEX-Ribo-Seq was demonstrated in primary neurons, revealing compartment-specific translatomes in dendrites, axons, and presynapses. Extending this approach in vivo, we applied APEX-Ribo-Seq to germ granules in Drosophila embryos, profiling the temporal transition from translationally silent mRNA storage to localized translation during early embryogenesis. Our results provide a comprehensive view of the spatiotemporal translatome and its regulation.

molecular biology↗

bioRxiv2025

eIF4G2-Mediated Translation Initiation of Histone Modifiers Is Essential for Intestinal Stem Cell Maintenance and Differentiation

eIF4G2 (also known as NAT1, p97, or DAP5) is an evolutionally conserved protein homologous to the C-terminal two-thirds portion of eukaryotic translation initiation factor (eIF) 4G. Despite its abundant and ubiquitous expression, the physiological and pathological functions of eIF4G2 are poorly understood. Here, we show that acute loss of eIF4G2 in adult mice results in rapid weight loss with abnormalities in multiple organs, including impaired maintenance and differentiation of intestinal stem cells. Genome-wide ribosome profiling revealed that eIF4G2 is critical for the translation of key histone modification proteins involved in intestinal stemness. Our study underscores the importance of eIF4G2-mediated translation initiation in multicellular organisms.

cell biology↗

bioRxiv2025

Mitochondrial translation termination, recycling, reinitiation, and rescue for in-frame and out-of-frame contexts

Because mitochondria diverged from a bacterial ancestor during evolution, the mitochondrial protein synthesis system includes both mRNAs and translation factors with unique characteristics. However, the molecular mechanisms underlying translation termination, recycling, and quality control remain unclear. Here, via high-resolution mitochondrial Ribo-Seq and Disome-Seq, we revealed: the specificity of release factors for different kinds of stop codons; the role of mtRF1 in vertebrates, which do not have noncanonical stop codons in their main ORFs; the recycling-coupled translation of internal ORFs; and the rescue of mitoribosomes in the early elongation stage. mtRF1L is a universal release factor that recognizes all stop codons, whereas mtRF1 recognizes only AGA/AGG noncanonical stop codons. Additionally, mtRF1 terminates the translation of out-of-frame ORFs that end with AGA/AGG. We also found that mtRRF and mtIF3 are required for mitoribosome recycling on stop codons and for the reinitiation of internal ORF translation. Mitoribosomes that stall at start codons are major substrates of the rescue factors ICT1, mtRF-R, and mtRES1. Moreover, HEMK1-mediated methylation of release factors enhances the termination reaction on stop codons. Our results provide insights into the mitoribosome dynamics that are associated with the completion of protein synthesis.

molecular biology↗

bioRxiv2024

Giant virus creates subcellular environment to overcome codon-tRNA mismatch

Codon usage consonant with the cellular tRNA pool is important for efficient translation. However, many eukaryotic viruses, including amoeba-infecting mimiviruses, have codon usage that is largely deviated from that of their host, despite using the host machinery for translation. This raises the question of how these viruses cope with the mismatch between tRNA supply and demand. Here we show that Acanthamoeba castellanii mimivirus generates a subcellular area in the host cells to translate virus mRNAs. A combination of genome-wide ribosome profiling and RNA sequencing showed that ribosomes traversed along viral mRNAs with fewer pausing events than were observed on amoeba mRNAs. Frequently used codons in viral mRNAs had higher tRNA accessibility than the same type of codons in amoeba mRNAs. tRNA sequencing showed that the tRNA pool was not greatly altered during the infection even though the virus encodes tRNA genes. Instead, by in situ labelling, we found that viral mRNAs and newly synthesized proteins were localized at the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation assists the virus in overcoming the mismatch between tRNA supply and demand.

microbiology↗

bioRxiv2024

Sense codon-misassociated eRF1 elicits widespread ribosome stalling and induction of quality control

The rescue pathway of stalled ribosomes and ribosome-associated quality control (RQC) serves as a surveillance system for aberrant translation that senses ribosome collisions. Although the molecular mechanism has been extensively studied, the endogenous contexts of ribosome collision rescued by the system are poorly understood. Here, beginning with a study of the codon specificity of the eukaryotic translation termination factor eRF1, we show that transient binding of eRF1 to the UUA sense codon leads to ribosome collision and provides a constitutive source of rescue substrates in humans. eRF1-selective Monosome-Seq and Disome-Seq revealed that eRF1 was recruited not only to stop codons but also to near-cognate sense codons, including the UUA codon. The eRF1 on UUA codons delays translation elongation but does not trigger the termination reaction. Remarkably, Disome-Seq with the depletion of ASCC3 and 4EHP, key factors for the ribosome rescue and translation initiation repression, showed that ribosomes stalled at UUA codons consist of a subpopulation suppressed by those factors. Failure of ribosome collision repression by ASCC3-4EHP triggers stress response, such as expression of the stress-induced transcription factor ATF3. This study highlights the impact of sense codon misrecognition by the termination factor on translation homeostasis in human cells.

molecular biology↗

bioRxiv2024

Synchronization between chloroplastic and cytosolic protein synthesis for photosynthesis complex assembly

Through symbiosis, subunits of chloroplastic complexes are encoded in distinct genomes in the nucleus and organelles. For plant cells to maintain the stoichiometry of subunits and respond to environmental cues, the orchestration of the nuclear and organellar gene expression systems is an essential task. However, the mechanism maintaining chloroplastic complexes remains largely enigmatic. Here, we simultaneously assessed the translatomes of the chloroplast and the cytoplasm via ribosome profiling and revealed the differential mechanisms employed by these two systems to cope with acute light/dark transitions: in chloroplasts, translational regulation is employed, whereas in the cytoplasm, control of the mRNA abundance is implemented. This strategy is widely conserved in land plants (Arabidopsis and the grass plant Brachypodium) and green algae (Chlamydomonas). The translational control in chloroplasts may be established based on organelle symbiosis; the primitive chloroplast in Glaucophyta (Cyanophora) was found to have already acquired translational control, whereas cyanobacteria (Synechocystis) control the mRNA abundance. Moreover, reduced plastoquinones and active cytosolic protein synthesis drive chloroplastic translation of the complex subunits in the light. Our work reveals an early origin of coordination of chloroplast and nuclear/cytoplasmic gene expression upon light exposure.

molecular biology↗

bioRxiv2024

Selective Translation Orchestrates Key Signaling Pathways in Primed Pluripotency

Pluripotent stem cell (PSC) identities, such as differentiation and infinite proliferation, have long been understood within the frameworks of transcription factor networks, epigenomes, and signal transduction, yet remain unclear and fragmented. Directing attention toward translational regulation, as a bridge between these events, promises to yield new insights into previously unexplained mechanisms. Our functional CRISPR interference screening-based approach revealed that EIF3D maintains primed pluripotency through selective translational regulation. The loss of EIF3D disrupts the balance of pluripotency-associated signaling pathways, impairing primed pluripotency. Moreover, we discovered that EIF3D ensures robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, selective translation by EIF3D tunes the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. Therefore, this study establishes a paradigm for selective translational regulation as a defining feature of primed PSC identity.

cell biology↗

bioRxiv2023

Chemical genetic interaction linking eIF5A hypusination and mitochondrial integrity

The eukaryotic translation factor eIF5A plays an important role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. eIF5A undergoes hypusination, a post-translational modification unique to this protein, although the role of hypusination in the function of eIF5A remains elusive. Here, we investigated the cellular defects induced by the hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Proteome, translatome, and transcriptome analyses indicated that GC7 downregulated a subset of mitochondrial proteins and DNA, causing mitochondrial stress and eliciting the integrated stress response. Chemical genomic screening using barcoded shRNA libraries identified genes encoding proteins involved in polyamine metabolism/transport and MPV17L2, a mitochondrial disease gene homologue whose product regulates mitochondrial translation. Depletion of MPV17L2 caused hypersensitivity to GC7 and phenocopied the effects of GC7 treatment. These results suggest that eIF5A hypusination and MPV17L2 synthetically regulate mitochondrial molecular synthesis and integrity.

cell biology↗