Biology subjects

Shichino, Y.

Publications and source records attributed to Shichino, Y..

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

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

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

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↗

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

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

Translation variation across genetic backgrounds reveals a post-transcriptional buffering signature in yeast

Gene expression is known to vary among individuals, and this variability can impact the phenotypic diversity observed in natural populations. While the transcriptome and proteome have been extensively studied, little is known about the translation process itself. Here, we therefore performed ribosome and transcriptomic profiling on a genetically and ecologically diverse set of natural isolates of the Saccharomyces cerevisiae yeast. Interestingly, we found that the Euclidean distances between each profile and the expression fold changes in each pairwise isolate comparison were higher at the transcriptomic level. This observation clearly indicates that the transcriptional variation observed in the different isolates is buffered through a phenomenon known as post-transcriptional buffering at the translation level. Furthermore, this phenomenon seemed to have a specific signature by preferentially affecting essential genes as well as genes involved in complex-forming proteins, and low transcribed genes. We also explored the translation of the S. cerevisiae pangenome and found that the accessory genes related to introgression events displayed similar transcription and translation levels as the core genome. By contrast, genes acquired through horizontal gene transfer events tended to be less efficiently translated. Together, our results highlight both the extent and signature of the post-transcriptional buffering.

systems biology↗

bioRxiv2023

Pateamine A mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only limited examples have been reported to date. Here, we show that pateamine A (PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, on GNG motifs. Through a systematic comparison of multiple eIF4A inhibitors by ribosome profiling, we found that PatA has unique mRNA selectivity in translation repression. Unbiased Bind-n-Seq revealed that PatA-targeted eIF4A exhibits a sequence preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. In silico simulation, combination of classical molecular dynamics simulation and quantum chemical calculation, and the subsequent development of an inactive PatA derivative revealed that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified DDX3, another DEAD-box protein, as an alternative target of PatA, showing the same effect as on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and mRNA-selective inhibition of protein synthesis by small-molecule compounds.

molecular biology↗

bioRxiv2023

Complexity and dynamics of in organello translation landscape assessed by high-resolution mitochondrial ribosome profiling

Since mitochondrial translation serves the essential subunits of the OXPHOS complex that produces ATP, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and revealed the intricate regulation of mammals in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at the anticodon stem loop in mt-tRNAs, including all possible modifications at the 34th position, by deleting the corresponding enzymes and harnessing patient-derived cells. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.

molecular biology↗

bioRxiv2023

No-go decay dampens zinc finger protein mRNAs in vertebrates

Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 downregulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. We further showed that mRNAs encoding C2H2-ZF were degraded in a ZNF598-dependent manner in HEK293T cells. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.

molecular biology↗

bioRxiv2023

Absolute calibration of ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to determine overall ribosome numbers in transcripts. Here, we overcame these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches measures the absolute number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method revealed the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy transforms ribosome profiling technique from relative to absolute quantification of translation.

molecular biology↗

bioRxiv2023

Translational regulation enhances distinction of cell types in the nervous system

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically-defined cells in the Drosophila brain, we here revealed substantial posttranscriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias towards the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open reading frames (uORFs) in the 5 leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

neuroscience↗

bioRxiv2023

CRISPRδ: dCas13-mediated translational repression for accurate gene silencing in mammalian cells

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)Cas13 systems, have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPRCas13). Thus, a more specific method of gene knockdown is needed. Here, we developed "CRISPR{delta}", an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent or internal ribosome entry site (IRES)-dependent translation. Strikingly, genome-wide ribosome profiling revealed the extremely high gene knockdown specificity of CRISPR{delta}. Moreover, fusion of a translational repressor to dCas13 ensured further improvement of the knockdown efficacy. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

molecular biology↗

bioRxiv2023

Gravitational and mechanical forces drive mitochondrial translation through the cell adhesion-FAK axis

Life on Earth has evolved in a form suitable for the gravitational force of 1 x g. Although the pivotal role of gravity in gene expression has been revealed by multiomics approaches in space-flown samples and astronauts, the molecular details of how mammalian cells harness gravity have remained unclear. Here, we showed that mitochondria utilize gravity to activate protein synthesis within the organelle. Genome-wide ribosome profiling revealed reduced mitochondrial translation in mammalian cells and Caenorhabditis elegans under both microgravity at the International Space Station and simulated microgravity in a 3D-clinostat on the ground. We found that attenuation of cell adhesion through laminin-integrin interactions causes the phenotype. The downstream signaling pathway including FAK, RAC1, PAK1, BAD, and Bcl-2 family proteins in the cytosol, and mitochondrial fatty acid synthesis (mtFAS) pathway in the matrix maintain mitochondrial translation at high level. Mechanistically, a decreased level of mitochondrial malonyl-CoA, which is consumed by activated mtFAS, leads to a reduction in the malonylation of the translational machinery and an increase in the initiation and elongation of in organello translation. Consistent with the role of integrin as a mechanosensor, we observed a decrease in mitochondrial translation via the minimization of mechanical stress in mouse skeletal muscle. Our work provides mechanistic insights into how cells convert gravitational and mechanical forces into translation in an energy-producing organelle.

molecular biology↗

bioRxiv2023

Thor-Ribo-Seq: ribosome profiling tailored for low input with RNA-dependent RNA amplification

molecular biology↗

bioRxiv2022

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier of antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocalgates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to Ophiocordyceps genus and its eIF4A, a molecular target of rocaglates, contains a amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered Colletotrichum orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

molecular biology↗