Biology subjects

Wakigawa, T.

Publications and source records attributed to Wakigawa, T..

7 recordsLinked to original sources

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

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

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↗

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↗

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

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↗

bioRxiv2022

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

molecular biology↗